Molecular basis of collagen galactosylation by GLT25D1

Domain: neuroscience
Published: 2026-02-06
Editorial outcome: major_revision

Abstract
GLT25D1 O-galactosylates hydroxylysine residues in collagen and is essential for collagen maturation and function. Dysfunctions of GLT25D1 cause various tissue disorders. Despite its biological significance, the action mechanism of GLT25D1 remains enigmatic. Here we report the cryo-EM structures of human GLT25D1 and its ternary complex with UDP and hydroxylated acceptor substrates, revealing a bi-lobed architecture for the GLT25D1 monomer that organizes into dimeric and hexameric oligomers. The N-lobe of GLT25D1 contains a high-affinity UDP-galactose binding site, and the C-lobe is the catalytic domain of the enzyme. The structures together with biochemical analyses unravel the key recognition of the consensus “Hyl-Gly” motif from collagen acceptor substrates and associated catalytic mechanism. We further demonstrate that GLT25D1 mutations linked to cerebral small vessel disease and musculoskeletal defects adversely affect its function via distinct mechanisms. Our findings elucidate the molecular mechanism underlying collagen glycosylation and provide a molecular framework for understanding GLT25D1-related diseases.

Introduction
Collagen, as the most abundant protein in all animal tissues and the primary constituent of the extracellular matrix (ECM), plays a crucial role in maintaining tissue structure, mechanical integrity, and cellular functions1–3. Its dysregulation is linked to numerous human diseases, including cancer, arthritis and more than 40 hereditary disorders. One defining feature of collagen is its O-linked glycosylation, a modification that critically regulates collagen properties and functions. During collagen biosynthesis, specific hydroxylysine (Hyl) residues are galactosylated by the enzyme GLT25D1 and GLT25D2 (also known as colgalt1 and colgalt2), and some of these modifications can be further glucosylated by lysyl hydroxylase 3 (LH3) (Fig. 1A)4–8. The resulting Gal(β1-O) monosaccharide and less abundant Glc(α1-2)Gal(β1-O) disaccharide are essential for proper collagen maturation and ECM-cell interactions9,10. The significance of collagen glycosylation is further underscored by its ubiquitous presence in all animal collagens and its high conservation across species, from sponges to humans11.

GLT25D1 catalyzes the initial step of collagen glycosylation and is broadly expressed across human tissues, while its homologous isoform, GLT25D2 (55% sequence identity), is restricted only to a few cell types4. GLT25D1 is indispensable for embryonic development and cell growth, typically in a collagen-dependent manner. Its inactivation in C. elegans causes growth defects and morphologic abnormalities12,13; simultaneous inactivation of both GLT25D1 and GLT25D2 impairs osteosarcoma cell viability, accompanied by intracellular accumulation of type I collagen14,15; GLT25D1 disruption in mice and zebrafish causes embryonic lethality, defects in vascular network formation or disorganized muscle fibers16,17. Beyond collagen, several additional substrates have recently been identified, including hepassocin and hepatocyte-derived fibrinogen-related protein 118, adiponectin19,20 and mannose-binding lectin21. GLT25D1 deficiencies have been linked to a spectrum of human diseases, including cerebral small vessel diseases (SVDs), vascular cognitive impairment (VCI), and connective tissue disorder17,22–24, highlighting its physiological importance.

GLT25D1, localized in the endoplasmic reticulum (ER), is responsible for transferring galactose from UDP-galactose to hydroxylysine (Hyl) at specific Yaa-positions within the characteristic Gly-Xaa-Yaa motifs of the collagen helical domain, where Yaa is often occupied by Hyl or Hyp (hydroxyproline)5–7. It contains two glycosyltransferase domains: a N-terminal CAZy GT2 domain and a C-terminal GT25 domain21. Mutagenesis analysis identified two DxD motifs (residues 166-168; residues 461-463) essential for GLT25D1 activities, located in the N-terminal and C-terminal domains, respectively25. Despite these findings, key mechanistic aspects of GLT25D1 – including its substrate recognition, catalytic mechanism, and even the precise location of its active site – remain elusive.

Here, we report the structures of apo-human GLT25D1 as well as its ternary complex with donor and acceptor substrates. Through structure-guided functional analyses, we elucidate the molecular basis of collagen galactosylation by GLT25D1 and provide mechanistic insights into the deleterious mutations of the enzyme that cause various collagen-related human diseases.

Results
Functional characterization of GLT25D1
Human GLT25D1 (30-619 Aa) was successfully expressed using the Pichia pastoris expression system, and it was further purified through immobilized metal affinity chromatography, followed by protease treatment to cleave the purification tag. Subsequent size-exclusion chromatography revealed two main elution peaks, indicating the presence of at least two oligomeric states of GLT25D1 in solution (Fig. 1B). To evaluate whether the recombinant protein was functional, we measured its collagen galactosyltransferase (ColGalT) activity using gelatin as the acceptor substrate. The activity was readily detected in the presence of donor substrate UDP-galactose and Mn2+, whereas no activity was observed with UDP-glucose and Mn2+, demonstrating donor specificity (Fig. 1C).

To further validate and quantitate the specific enzymatic activity of GLT25D1, we selected a native peptide “GAKGEKG” in collagen alpha-1(VII) chain (Uniprot ID: Q02388), which contains two Gly-Xaa-Lys tripeptides (“GAK” and “GEK”) populated in different collagen chains11. We synthesized this designed acceptor peptide SGA-Hyl-GEKGS (referred to as “hydroxylated acceptor peptide” in this study), where hydroxylysine (Hyl, underlined) was positioned next to an alanine residue. Activity assay using this hydroxylated acceptor peptide confirmed the donor preference for UDP-galactose, consistent with the results obtained with the gelatin acceptor (Fig. 1C). In contrast, GLT25D1 exhibited no apparent activity with a control peptide lacking hydroxylysine (Fig. 1C). Utilizing the designed peptide, the reaction product by GLT25D1 was analyzed via UPLC-MS analysis. When GLT25D1 was incubated with UDP-glucose or UDP-galactose, only UDP-galactose can introduce an expected molecular weight increase of 162 Da to the peptide, confirming galactose modification (Fig. 1D, E). Simultaneous incubation with both UDP-galactose and UDP-glucose resulted only in monosaccharide modification (Fig. 1F). These findings are consistent with a previous study4, unequivocally demonstrating that GLT25D1 exclusively possesses galactosyltransferase activity when modifying hydroxylysine in collagen.

Oligomeric GLT25D1 and its overall structure
To gain deeper insights into the molecular mechanism of GLT25D1, we conducted a cryo-electron microscopy (cryo-EM) study (Supplementary Fig. 1 and Supplementary Table 1). The protein sample analyzed was prepared by pooling fractions corresponding to the two major oligomeric peaks isolated by size-exclusion chromatography (Fig. 1B). 2D classification analysis revealed that GLT25D1 exists in two oligomeric states as dimer and hexamer (Fig. 2A), consistent with size-exclusion chromatography analysis (Fig. 1B). 3D reconstruction of the hexameric state allowed us to derive a 3.1-Å resolution map of six GLT25D1 protomers arranged as a trimer of three dimers (designated as P1-P1’; P2-P2’; P3-P3’) (Fig. 2B). This hexamer measures approximately 110 Å in height and 120-150 Å in diameter and has a C3 symmetry axis coinciding with a central axial channel approximately 19 Å wide (Fig. 2B). Further symmetry expansion and local refinement with dimer mask yielded a 3.3-Å resolution map, showing improved density quality of one composite dimer (Supplementary Fig. 1C, E, F). This enabled the de novo building of the atomic model of GLT25D1 homohexamer, with most residues resolved except for one unstructured region (residues 561-619).

Each composite monomer of the GLT25D1 hexamer exhibits a bi-lobed architecture, with both domains adopting the canonical GT-A fold characteristic of glycosyltransferase (Fig. 2C, D)26. The N-terminal glycosyltransferase domain (NGT; residues 51-328) features a central β-sheet (β1-7) flanked by multiple α-helices, forming a Rossmann-like fold topology (Supplementary Fig. 2). Likewise, the C-terminal glycosyltransferase domain (CGT; residues 332-561) is organized around a central six-stranded β-sheet (β8-13) encompassed by several α-helices (Supplementary Fig. 2). The interface between two glycosyltransferase domains (NGT and CGT domains) is mediated by an extended linker (M-loop, residues 328-340). This is further strengthened by multiple interactions among α7, α8, α11, and the loop preceding α11, including the polar contacts (E314 with R439; E306 with R446) and hydrophobic packing among M309, H310, L313, M316, A364, F440 and I442 (Fig. 2D).

Molecular basis of GLT25D1 oligomeric assembly
Our results showed that GLT25D1 predominantly exists as dimers and hexamers (Fig. 2A), with the hexamer arranged as a trimer of three dimers (Fig. 2E and Supplementary Fig. 3A–C), suggesting that the dimer is the basic unit for GLT25D1 oligomerization. This dimer is formed by interactions at the NGT domains (“head”) of both protomers, adopting a butterfly-like shape with two-fold symmetry (Fig. 2F). Each “wing” of the head-to-head dimer extends outward from the central interface, with the two outer C-terminal CGT domains spatially separated by approximately 165 Å. The central interface is extensive with a buried surface area of 2942 Å2, involving multiple loops of the NGT domain organized into three layers (Fig. 2G). In the top layer, the two protomers exchange their extended N-terminal loops (N-loop, residues 38-51), where residues S47 and P48 of one protomer forms hydrogen bonds with consecutive alanine residues (A245-A246) of the neighboring protomer. In the middle layer, the close stacking of arginine pairing (R53-R53) is further reinforced by a charged interaction between R53 and D160. In the bottom layer, W158 strategically inserts itself into the space between R53 and H113, establishing robust cation-pi and pi-pi interactions that are likely critical for dimerization. Indeed, this is strongly corroborated by the observation that H113A/W158A double mutations resulted in a pronounced shift in the oligomer equilibrium, predominantly favoring monomer formation in solution (Fig. 2H). Interestingly, the galactosyltransferase activity of the H113A-W158A mutant against gelatin was not dramatically affected relative to that of wild-type GLT25D1 (Fig. 2I), suggesting that the oligomerization is not required for at least in vitro catalytic activity.

In the GLT25D1 hexamer, three dimer units are arranged around the three-fold axis (Fig. 2E). This configuration symmetrically assembles C3-related protomers (P1, P2 and P3; P1’, P2’ and P3’) into two stacked and non-equivalent trimer rings, with the bottom ring (P1’, P2’ and P3’) extending further away from the C3 axis. The hexamer is stabilized by crossover contacts of two neighboring dimer units (e.g., P1-P1’ and P3-P3’) and this forms two inter-dimer interacting interfaces (I and II) located exclusively within their NGT domains near the intersection, burying a total surface area of 1924 Å2 (Supplementary Fig. 3D). Specifically, at ‘Interface I’ between the interacting dimers (e.g., P1-P1’ and P3-P3’), W43 on N-terminal loop of protomer P3’ stacks with R223 in the NGT domain of the neighboring protomer P1, which was further strengthened by an interprotomer salt-bridge interaction between E286 and R326 (Supplementary Fig. 3E). At ‘Interface II’, W43 on N-terminal loop of protomer P3 stacks with A245, A246 and R248 in NGT of the adjacent protomer, reinforced by a polar interaction between R42 and N249 (Supplementary Fig. 3F).

Identification of UDP-galactose endogenously bound to NGT domain
Both NGT and CGT have been implicated in catalyzing collagen galactosylation25. Notably, despite no donor ligand being added during sample preparation, densities consistent with a UDP-hexose ligand were observed in an open pocket of the NGT domain (Fig. 3A, B). To identify the co-purified endogenous ligand, we employed LC-ESI-MS/MS analysis. The mass data from the extracts of purified GLT25D1 matched either UDP-galactose or its isomer, UDP-glucose (Supplementary Table 2). Furthermore, the elution profile aligned with UDP-galactose standard, rather than UDP-glucose (Fig. 3C). Consequently, we modeled UDP-galactose, fitting well into the ligand cryo-EM densities (Fig. 3B). These findings strongly suggest the tight and specific binding of UDP-galactose to the NGT domain of GLT25D1. Indeed, in our model, the UDP-galactose is held in place by a rich network of interactions, forming a highly conserved cavity suited for its accommodation (Fig. 3B and Supplementary Fig. 4). Specifically, the uridine moiety is sandwiched between R61 and V143 and further stabilized by hydrogen bonds with Y126 side chain and L59 main chain. The diphosphate moiety is stabilized by K133 and an Mn2+ ion, where the ion was coordinated by the DXD motif (D166 and D168). Additionally, the galactose moiety is packed with W135 and engaged with a series of polar contacts with surrounding R147, D265 and H235.

Surprisingly, mutational analysis did not support this UDP-galactose binding site in the NGT domain as the ColGalT catalytic site. We generated GLT25D1 mutants with alanine substitutions in the NGT domain, targeting residues involved in uridine stabilization (R61, V143), ion coordination (D166, D168), and galactose interaction (W135, R147, H235, D265). Among the subset of mutants that could be successfully purified (Supplementary Fig. 5; see discussion for explanations), their residual activities varied considerably, ranging from wild-type levels (R147A) to minor reductions (D265A, W135A), with only D166A exhibiting the most substantial decrease in catalytic activity (Fig. 3D). Collectively, these findings strongly suggest that the ColGalT catalytic site resides outside the NGT domain.

Ternary complex structure of GLT25D1 with bound substrates
To localize the ColGalT catalytic site, we expressed and purified the isolated NGT domain (residues 30-333) and CGT domain (residues 332-619) of GLT25D1 and assessed their individual catalytic activities through two methods. First, in assays monitoring UDP generation, the NGT domain exhibited no enzymatic activity, whereas the CGT domain readily showed activity with both gelatin and hydroxylated peptide substrates (Fig. 4A). Second, mass spectrometric reaction product analysis in the presence of hydroxylated peptide substrate confirmed no modification by the NGT domain but significant modification by the CGT domain (Supplementary Fig. 5H). These results indicate that the ColGalT catalytic site resides within the CGT domain, consistent with our mutational analysis of the NGT domain (Fig. 3D).

To investigate the molecular basis of substrate recognition and catalysis, we incubated GLT25D1 (pooled from the 11.8 mL SEC peak in Fig. 1B) with UDP-galactose, hydroxylated peptide acceptor (as that tested in Fig. 1D) as well as Mn2+, and performed cryoEM analysis (Supplementary Fig. 6). The resulting complex structure retained the UDP-galactose:Mn2+ in the NGT domain as observed in the apo enzyme, but revealed additional densities for UDP:Mn2+ and acceptor peptide in the CGT domain, clearly confirming the CGT domain as the ColGalT catalytic domain (Fig. 4B and Supplementary Figs. 7 and 8). Notably, comparing the substrate-bound and apo states of the CGT domain revealed the ordering of a 20-residue loop (Y561-E580; termed the ‘lid loop’) near the CGT active site, which is disordered in the apo structure (Fig. 4C, Supplementary Fig. 7A, B). Two highly conserved residues, H560 and R351, function to stabilize this ordered loop through backbone stacking at its beginning and a salt bridge with D570 at its middle, respectively (Supplementary Fig. 7C). Alanine substitutions of either residue compromised enzymatic activity, underscoring the functional importance of this loop (Supplementary Fig. 7C, D). Indeed, enclosed by this rearranged lid loop, UDP is bound within an embedded pocket of the CGT domain, lined with a series of conserved residues (Fig. 4D and Supplementary Fig. 9). Specifically, the uracil moiety of UDP is held in position by a hydrogen bond to G377 main chain and by stacking between C412 and L348 while the ribose moiety forms hydrogen bonds with D436. The pyrophosphate moiety is stabilized by a Mn2+ metal ion, coordinated by D437. Additional stabilization of pyrophosphate moiety comes from contacts with R354 and residues S569 and T571 from the lid loop. Substituting these ligand-binding site residues individually to alanine markedly reduced enzyme activity (Fig. 4E). In particular, D436A, D437A, S569A and T571A nearly abolished activity (Fig. 4E).

Acceptor peptide binding mode and catalytic mechanism
Collagens feature a (Gly-Xaa-Yaa) repeating pattern and thereby GLT25D1 specifically galacosylates hydroxylysine (Hyl) residues within the “Xaa-Hyl-Gly” consensus sequence6. This is evident from the sequence analysis of confirmed galactosylation sites in human collagen type II (Fig. 5A)27. Structural analysis of the ternary complex (GLT25D1/acceptor peptide/UDP) uncovered the molecular basis for the recognition of this consensus sequence. In the CGT active site, EM densities corresponding to a strand of four residues were identified, enabling the modeling of an acceptor peptide “Ala-Hyl-Gyl-Glu” (Fig. 5B, C). The acceptor peptide interacts with GLT25D1 primarily via its amide backbone, mediated by conserved GLT25D1 residues on both sides of the peptide. Specifically, K470, Y494, and W495 form one side of the binding interface, while Y561 and T562 define the opposite side (Fig. 5B). These residues form a cleft connecting the binding sites of acceptor and donor substrates. Notably, the narrowest constriction of the cleft, measuring 6.8 Å in width, is defined by K470 and T562, with the β-carbon of T562 positioned merely 4.1 Å away from the α-carbon of glycine (at subsite +1) of the acceptor peptide (Fig. 5D). This geometry specifically accommodates the invariant glycine residue following Hyl, while excluding bulkier residues. Furthermore, the invariant hydroxylysine (Hyl) is stabilized within this cleft by the “YW” motif (Y494 and W495), with its ε-amino group potentially anchored by E523 (Fig. 5B). This configuration positions the 5-hydroxyl group of hydroxylysine in close proximity to the β-phosphate of UDP in CGT active site, appropriate for galactose transfer. Indeed, mutagenesis experiments support these structural insights: the W495A mutation abolished enzymatic activity; K470A, Y494A, Y561A and E523A mutations markedly reduced GLT25D1 activity (Fig. 5E). Interestingly, alanine substitution of T562 preserved activity (Fig. 5E), suggesting that its interaction with acceptor peptide may rely more on the β-carbon of this residue (Fig. 5D).

The structure of the ternary complex also provides valuable insights into the catalytic mechanism of GLT25D1. Inspection of the active site within the CGT domain revealed that D522, an invariant residue conserved from humans to sponges, is located within 4 Å of the acceptor hydroxyl group (Fig. 5B and Supplementary Fig. 4). This proximity ideally positions D522 as the catalytic base, primed to activate the nucleophile, consistent with an SN2-type inverting catalytic mechanism (Fig. 5F)26. Similar mechanisms have been observed in other glycosyltransferases, such as POMGnT1, POGLUT1 and POFUT2, whereas aspartate or glutamate serves as a general base28–30. Indeed, site-directed mutagenesis of D522 completely abrogated enzymatic activity, underscoring its essential role in catalysis, as demonstrated by both UDP generation assays (Fig. 5E) and mass spectrometric analysis of reaction products (Supplementary Fig. 5I). Furthermore, we observed that the ColGalT activity requires Mn2+ for both the full-length GLT25D1 and its truncated CGT domain (Supplementary Fig. 5J, K), consistent with previous findings31–33. This highlights the critical role of Mn2+ in catalysis, likely by facilitating the departure of the diphosphate leaving group, as proposed for other glycosyltransferases26. Collectively, these structural and functional analyses conclusively define the active site of GLT25D1 and identify its key residues involved in collagen galactosylation (Figs. 4 and 5).

Molecular insights into pathogenic mutations of GLT25D1
Deficiencies in GLT25D1 have been linked to a spectrum of human diseases, with several potential pathogenic mutations identified in cerebral small vessel disease (SVD; L151R, A154P, E366Rfs, G377R, G209fs, and G209_K212del), vascular cognitive impairment (VCI; R471W) and musculoskeletal defects (W135R)17,22–24. To elucidate the mechanistic impacts of these mutations, we mapped them onto the GLT25D1 ternary complex structure (Fig. 6A). This analysis revealed that the SVD-related frameshift mutations (E366Rfs and G209fs) result in truncated GLT25D1 proteins lacking the catalytic CGT domains, likely rendering the enzyme inactive (Fig. 2C). Additionally, the SVD-associated point mutations L151R and A154P, located in helix α3, are expected to disrupt the domain’s core fold stability (Fig. 6A). Specifically, the L151R mutation introduces a large, charged residue into a hydrophobic core, while A154P is likely to impair helical structure and packing. These destabilizing effects are consistent with the dramatically reduced expression levels observed for both mutants (Fig. 6B), in agreement with a previous study22. Further analysis revealed that SVD mutant G209_K212del and musculoskeletal-defect-related mutant W135R are situated near the bound UDP-galactose in the NGT domain (Fig. 6A). Functional assays demonstrated that G209_K212del preserves half the normal activity, whereas W135R almost abolishes the enzymatic function (Fig. 6C). Lastly, the SVD-associated mutation G377R and VCI-related mutation R471W are located within the active site of CGT domain (Fig. 6A). Residue G377 is directly involved in donor ligand binding (Fig. 4D), while R471 sits next to the critical acceptor-binding residue K470 (Fig. 5B). Indeed, neither G377R nor R471W appeared to markedly affect protein expression levels (Fig. 6B), but G377R almost eliminates GLT25D1 activity and R471W leads to a substantial reduction in enzymatic function (Fig. 6C).

Dysfunctions of collagen and its glycosylation have increasingly been recognized as critical factors in cancer progression and metastasis10,34,35. Motivated by this association, we identified cancer-associated GLT25D1 mutations and analyzed them by mapping onto the ternary complex structure. These mutations are distributed across both the NGT and CGT domains (Supplementary Fig. 10), consistent with the functional importance of each domain. Notably, four cancer-associated mutations (R147H, R147C, D168H, and G377D) are situated at key ligand-binding interfaces and are likely to impair GLT25D1 function: R147H, R147C, and D168H in the NGT domain (Fig. 3B), and G377D in the CGT domain (Fig. 4D). Moreover, GLT25D1 alterations have been linked to tumorigenesis in several cancer types, including malignant mammary tumors and kidney renal clear cell carcinoma36,37. These findings highlight the broader implications of GLT25D1 dysfunction, extending its relevance to cancer biology.

Methods
Expression and purification of human GLT25D1
The gene encoding human GLT25D1 (GenBank ID: NM_024656.4) was amplified from the cDNA of 293T cells. The secreted form of GLT25D1, encompassing residues 30-619, was cloned into the pPICZ C vector (Thermo Fisher Scientific) with a C-terminal HRV3C protease recognition site followed by a 10×His tag. Site-directed mutagenesis was performed using an inverse PCR strategy45, with the plasmid harboring the wild-type GLT25D1 gene as the template and corresponding mutation-specific primers. The resulting PCR products were treated with DpnI (NEB; 37°C, 1h) to digest the methylated template, phosphorylated with T4 polynucleotide kinase (NEB; 37°C, 30min), and ligated using T4 DNA ligase (NEB; 16°C, 16h). The circularized DNA was then transformed into E. coli DH5α competent cells (Sangon Biotech). Clonies with related mutations were verified by PCR and confirmed by DNA sequencing.

For expression, the recombinant plasmids were linearized by restriction enzyme PmeI and subsequently transformed into Pichia pastoris X33 (Thermo Fisher Scientific, C18000) competent cells via electroporation. Transformants were sequentially inoculated in YPD and BMGY media at 28°C. When OD₆₀₀ reached 2-6, cells were harvested by centrifugation at 300×g for 5min. The BMGY medium was then removed, and the cell pellet was resuspended in BMMY medium. Protein expression was induced at 24°C for 72h, with 0.5% (v/v) methanol added every 24h, following Pichia Expression Kit USER GUIDE (Invitrogen, Publication Number MAN0000012).

For protein purification, the collected cells were lysed in buffer A (25mM HEPES/NaOH, 500mM NaCl, pH 8.0). The supernatant was collected by centrifugation at 15,000×g for 30min. Protein purification was performed using an ÄKTA pure chromatography system equipped with a 5 mL column of nickel nitrilotriacetic acid (Ni-NTA) resin (GE Healthcare). The column was sequentially washed with 10 column volumes each of buffer A containing 20mM, 50mM, and 100mM imidazole, respectively. The target proteins were eluted with buffer A supplemented with 500mM imidazole. To remove the C-terminal 10×His tag, the eluted protein was digested with HRV3C protease (Genscript) at a 1:20 (w/w) protease-to-protein ratio, followed by overnight incubation at 4°C. The digested protein was desalted with a HiPrep™ 26/10 column (GE Healthcare), concentrated and further purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated in buffer A. The collected fractions were concentrated to 1–2 mg/mL for cryo-EM grid preparation. Protein concentration was quantified by A280 using NanoDrop Spectrophotometers, with an extinction coefficient of 107635 M–1cm–1 and a molecular weight of 68.3 kDa, respectively.

For activity analysis, wild-type and mutant proteins were purified by affinity chromatography as described above. The purified proteins were then desalted and exchanged into buffer A (25mM HEPES/NaOH, 500mM NaCl, pH 8.0) using a HiPrep™ 26/10 column (GE Healthcare) and concentrated to 0.5 mg mL⁻¹ for subsequent assays.

Glycosyltransferase activity assays
Glycosyltransferase activity was assayed using the UDP-GloTM Glycosyltransferase Assay (Promega). The hydroxylated peptide SGA-Hyl-GEKGS (where Hyl denotes hydroxylysine) was sourced from Shenzhen PepBiotic. Its synthesis involved the prior preparation of the hydroxylysine building block, which was subsequently incorporated into the peptide sequence via solid-phase peptide synthesis (SPPS)46. The corresponding non-hydroxylated peptide SGA-K-GEKGS was obtained from GenScript Biotech. The standard reaction mixture (25µL) contained 0.625mM peptide, 0.7 µM (0.05 mg/ml) of wild-type or mutated GLT25D1 and a reaction buffer containing 50mM Tris-HCl, pH 7.5, 150mM NaCl, 0.1mM MnCl2. The reaction was initiated by adding UDP-galactose or UDP-glucose (Promega) to a final concentration of 0.5mM. The reaction was carried out at 37°C for 1h. For assays utilizing gelatin as the substrate, 1.5 mg/mL gelatin (solubilized through heating denaturation at 95°C for 10min) was added to the reaction mixture, according to a previous study47. After the reaction, the enzymatic reaction mixture was mixed with an equal volume of UDP-GloTM Detection Reagent (15μL) in white polystyrene, 384-well assay plates (Perkin-Elmer). The plates were incubated at 25°C for 1h, and luminescence was recorded using a Pherastar FS system (BMG Labtech). To ensure accurate activity comparisons across variants, SDS-PAGE band intensity quantification was used to normalize the enzyme amounts for WT GLT25D1 and its mutants. All experiments were performed in triplicate. Uncoupled control reactions were under identical conditions but in the absence of the acceptor substrate. The wild-type (WT) activity against gelatin is normalized to WT activity against the hydroxylysine peptide. For kinetic analysis, the reaction contained 0.7 μM of GLT25D1 or its variants, 500 μM of the UDP-Gal, and gelatin at concentrations ranging from 0.5 mg/mL to 30 mg/mL. After incubation at 37°C for 5min, the reactions were analyzed using the UDP-Glo luminescence assay (Promega) as described above. Kinetic parameters (Km, kcat, and kcat/Km) and their standard errors were derived by fitting the data to the Michaelis–Menten equation via nonlinear regression in GraphPad Prism 8. All experiments were performed in triplicate.

LC-MS analysis of enzymatic products
For LC-MS analysis of enzymatic products, enzymatic reactions were prepared by combining 1.7 µM GLT25D1 or its mutant variants with 1.25mM hydroxylated peptide substrate, and 0.5mM either UDP-Galactose or UDP-Glucose in a reaction buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.1mM MnCl2. The reactions were carried out at 37°C for 3h. Following the enzymatic reaction, each sample was concentrated by nitrogen blow-down evaporation and subsequently reconstituted in a methanol/water (80:20, v/v) solution. The samples were then centrifuged at 15,000×g for 20min at 4°C, and the resulting supernatants were analyzed using a UHPLC-MS system following a previous study48,49. The analysis was performed using a Vanquish UHPLC system (Thermo Scientific) coupled to Q-ExactiveTM Plus mass spectrometer (Thermo Scientific). A Hypersil Gold Vanquish column (Thermo Scientific, 2.1×100mm, 1.9 µm) was used for chromatographic separation, with a gradient mobile phase of solvent A (0.1% formic acid in water) and solvent B (acetonitrile) at a flow rate of 0.3 mL/min. The gradient elution conditions were as follows: 10% B (0−2min), linear increase to 50% B (2–10min), 80% B (10–13min), 95% B (13–14min), and return to 10% B (14–18min). Mass spectrometric detection was conducted in positive ion mode with a spray voltage of 3.5 kV and a heated capillary temperature of 320°C. MS scans were performed over ranges of m/z 70-1500, with a resolution of 70,000, an automatic gain control (AGC) target of 1×106 and a maximum injection time of 100ms. The MS experiments were run once per sample.

Cryo-EM data acquisition
For single-particle cryo-EM analysis, the grids (Quantifoil R1.2/1.3 Au, 300 mesh) were glow-discharged using a PELCO EasiGlow unit at (15mA for 60s). Then a 3 µL aliquot of purified GLT25D1 (pooled from the 10.3-ml and 11.8-mL SEC peak in Fig. 1B) at a concentration of 1.2 mg/mL was applied to glow-discharged grids. The grids were blotted for 3–4 s at 100% humidity and flash-frozen in liquid ethane using a FEI Vitrobot Mark IV System (Thermo Fisher Scientific). For the ternary complex sample, GLT25D1 (1.2 mg/mL; pooled from the 11.8-ml SEC peak in Fig. 1B) was mixed with 0.8mM UDP-galactose and 1mM hydroxylated acceptor peptide, supplemented with 1mM MnCl2. The mixture was incubated at 4°C for 30min before freezing the cryo-EM grids as described above.

Cryo-EM data were collected on a FEI Titan Krios electron microscopy operated at 300kV equipped with a Gatan K3 Summit camera and a GIF Quantum energy filter (slit width 20 eV). Automated data acquisition was performed with FEI EPU. Micrographs were recorded under super-resolution counting mode in a physical pixel size of 1.076 Å for GLT25D1 sample and 0.855 Å for ternary complex sample. Defocus values varied from −1.2 μm to −3 μm. A total exposure of 1.3 s was dose-fractionated into 32 frames, yielding a total accumulated fluence of 50 electrons per square angstrom.

Image processing and 3D reconstruction
The dose-fractionated movies collected were motion-corrected and dose-weighted with MotionCor250. The contrast transfer function (CTF) parameters for individual micrographs were determined using CTFFIND451. Micrographs displaying poor quality, as assessed by manual inspection, were excluded from further analysis.

For the GLT25D1 dataset, the image-processing steps were performed using RELION-352, and processing workflow is presented in Supplementary Fig. 1. A set of 1500 particles was manually selected to generate 2D class templates for reference-based automatic particle picking. The automatic picking yielded in 773,881 particles from 1192 micrographs. Two rounds of reference-free 2D classification were performed to remove contaminants and low-quality particles, resulting in a cleaned set of 720,616 particles. 3D classification was performed using an ab initio map generated by RELION-3 as the initial reference model. The 3D class displaying clear hexamer features (125,162 particles) was selected. Subsequent refinement with C3 symmetry, combined with CTF refinement and polishing, resulted in a map with an overall resolution of 3.1 Å. This reconstruction revealed weak density within the outer C-terminal domain (CTD) regions, suggesting resolution loss due to symmetry averaging. To address this, we expanded the C3-symmetry-imposed dataset (125,162 particles) with the symmetry expansion tool in RELION, increasing particle count to 375,486. Subsequently, focused refinement was performed on this expanded dataset, using a focused mask encompassing a single GLT25D1 dimer. This strategy generated a map with significantly improved density in both NTD and CTD domains, achieving an overall resolution of 3.3 Å.

For the ternary complex dataset, image processing was performed using a combination of cryoSPARC53 and RELION-352. The corresponding workflow is provided in Supplementary Fig. 6. Initially, a total of 13,403,413 particles were automatically picked from 14,079 micrographs, using a blob-based particle picking algorithm in cryoSPARC. Multiple rounds of 2D classification were performed, and class averages displaying recognizable secondary structures were selected. The resulting subset (3,033,718 particles) was subject to multi-class ab initio reconstruction followed by heterogeneous refinement. The dominant class (1,896,674 particles) with high-resolution features was selected and imported into RELION-3 for further processing. Subsequent refinement with C2 symmetry yielded a map with an overall resolution of 2.9 Å. We noted the outer C-terminal domain (CTD) exhibiting relatively weak density due to an elongated dimer shape, which hindered the precise identification of bound substrates. To address this, we expanded the C2-symmetry-imposed dataset using the symmetry expansion tool in RELION. The expanded set (3,793,348 particles) underwent one round of 3D classification without alignment, using a focused mask around a single GLT25D1 subunit. A 3D class displaying the best density features in the CTD region was selected for 3D refinement with a focused monomer mask, resulting in a map of the ternary complex at an overall resolution of 3.2 Å.

For both datasets, the overall resolutions were estimated based on the gold-standard Fourier shell correlation 0.143 criterion54. Local resolution distribution was estimated using ResMap55.

Model building and refinement
The GLT25D1 dimer was initially built from the map refined with a focused dimer mask (GLT25D1 dataset; Supplementary Fig. 1). The initial monomeric model was generated using AlphaFold356 and two copies of this model were fitted into the EM densities as rigid bodies in Chimera to generate a preliminary dimer model57. It was subsequently improved by interactive rebuilding in Coot58 and iterative refinement in PHENIX with secondary structure and geometry restraints applied59. For the hexameric GLT25D1 structure, three copies of the GLT25D1 dimer were docked into the consensus map refined with C3 symmetry (GLT25D1 dataset; Supplementary Fig. 1). Rigid-body adjustments of each monomer were performed in Chimera to slightly optimize the fit. The ternary complex structure was built based on the map refined with a focused monomer mask (ternary complex dataset; Supplementary Fig. 6).

The final models were validated using MOLPROBITY60. The illustrated figures were prepared using PyMOL (Schrödinger, LLC)61, Chimera and ChimeraX57,62. Detailed statistics for 3D reconstructions and model refinements are provided in Supplementary Table 1.

Identification of the endogeneously bound ligand in GLT25D1
Purified GLT25D1 protein was digested overnight with trypsin at 30°C. For ligand extraction, four volumes of ice-cold HPLC-grade methanol (Thermo Fisher Scientific) were added to the digested sample, followed by thorough mixing and incubation on crushed ice for 20min. The crude extracts were then centrifuged at 13,000×g for 15min at 4°C. The supernatant was collected, evaporated under vacuum and resuspended in 200 µL of Milli-Q water. Detection and identification of the analytes were conducted with UPLC-MS/MS method63,64. Chromatographic separation was performed on an ACQUITY UPLC H-Class plus system (Waters, Wilmslow, UK) equipped with a BEH amide column (2.1×100mm, 1.7 µm; Waters, Wilmslow, UK). An injection volume of 5 µL was used, and the mobile phase consisted of solvent A (50mM ammonium formate solution, pH = 3.6) and solvent B (acetonitrile) at a flow rate of 0.4 mL/min. The elution was maintained at (79% solvent A):(21% solvent B). Ultra-pure UDP-sugar standards (Promega) were used for calibration. Mass spectrometric analysis was conducted in negative ion mode on a TQ-XS system (Waters, Wilmslow, UK) equipped with an electrospray ionization (ESI) source. Optimal multiple reaction monitoring (MRM) conditions were established for analyte detection. Two individual transitions were monitored for each analyte under the following parameters: ion spray voltage, 3.5 kV; auxiliary gas pressure, 5 arbitrary units; ion transfer tube temperature, 350°C; ion source temperature, 150°C. Detailed values of collision energy, cone voltage, and transitions for the MRM mode are provided in Supplementary Table 2.

Differential scanning fluorimetry
The thermostability of wild-type and GLT25D1 mutants was evaluated using differential scanning fluorimetry65. For each assay (20 µL in total volume) prepared in a 96-well PCR plate, 2 µg of purified protein samples were mixed with 5× SYPRO Orange (Sigma Aldrich; 1:1000 dilution of the stock) in buffer (25mM HEPES, pH 8.0, 500mM NaCl). Thermal denaturation profiles were determined by monitoring fluorescence over a temperature gradient on a QuantStudioTM 3 Real-Time PCR Instrument (Applied Biosystems). The incubation was started at 25°C for 2min, followed by a linear temperature ramp from 25°C to 90°C at a rate of 1°C per min. Melting curve analysis was performed using Protein Thermal Shift Software v1.4 (Applied Biosystems). The melting temperature (Tm) was calculated from the inflection point of the melting curve (derivative melting point). All experiments were conducted in quadruplicate, and the mean values with the standard deviation of the respective Tm values were presented.

Western blots
Wild-type and mutated GLT25D1 constructs were generated and expressed in Pichia cells as described above. Briefly, transformed yeast cells were induced in BMMY medium containing 0.5% (v/v) methanol for 24h. Cells were then harvested, lysed, and equal amounts of protein were separated on 12% SDS-PAGE gels and electroblotted onto a nitrocellulose membrane (Milipore Sigma). For immunodetection, the membrane was probed with a mouse anti-GLT25D1 primary antibody (1:1000 dilution; cat no. 16768-1-AP, Proteintech) followed by incubation with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (1:5000 dilution; cat no. SA00001-1, Proteintech). GAPDH level was used as a loading control for all samples66, and was detected with a rabbit anti-GAPDH primary antibody (1:5000 dilution; cat no. 10494-1-AP, Proteintech) and an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution; cat no. SA00001-2, Proteintech). Protein bands were visualized using the Odyssey infrared imaging system (LI-COR Biosciences), and densitometric analysis was performed to quantify protein expression levels. The uncropped and unprocessed scans are provided in the Source Data file.

Molecular dynamics simulations
Molecular dynamics (MD) simulations were carried out using GLT25D1 structure, both with and without UDP-Galactose molecule bound to the NGT domain. Force field parameters for GDU were generated using Antechamber. The simulations were performed with the Amber 20 software package, employing the ff19SB force field for the protein and the TIP3P water model. System preparation was conducted using the LEaP module, where the protein–ligand complex was solvated in a truncated octahedral water box with a minimum buffer distance of 12 Å from the solute to the box edge. Sodium and chloride ions were added to neutralize the system and adjust the ionic strength to ~150mM.

Energy minimization was performed in two stages: first, with positional restraints on the solute using the conjugate gradient algorithm for 2000 steps, followed by an unrestrained minimization of the entire system for 2500 steps (1000 steps of steepest descent and 1500 steps of conjugate gradient). The minimized system was then gradually heated from 0 to 300 K over 100 ps under constant-volume (NVT) conditions, with weak restraints on the solute. Finally, 100 ns production MD simulations were conducted under constant-pressure (NPT) conditions using the pmemd.CUDA engine in Amber 20. All simulations were performed with four independent replicates.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript presents a detailed structural and mechanistic study of human GLT25D1, an essential collagen O-galactosyltransferase. Using cryo-EM, the authors resolve the architecture of GLT25D1, demonstrating a two-lobed structure in which the N-lobe binds UDP-Gal non-catalytically, while the C-lobe contains the catalytic CGT domain. Importantly, the study shows that GLT25D1 predominantly forms oligomers, a finding that may have important implications for its structural stability, regulation, or broader cellular function, although dimerization is not essential for enzymatic activity. The authors further integrate biochemical assays and mutational analysis to elucidate substrate recognition and the effects of disease-related mutations.\nOverall, this study makes a valuable contribution to our understanding of collagen glycosylation and opens new mechanistic avenues for further research.\nMajor Points:\nThe observation that the non-catalytic N-lobe is critical for enzymatic activity is particularly intriguing and suggests a possible mechanism of allosteric regulation. To clarify this aspect:\n• Perform steady-state kinetic analyses (Km and kcat) for both UDP-Gal and peptide substrates, comparing wild-type GLT25D1 with N-lobe mutants (e.g., D166A).\n• Use molecular dynamics (MD) simulations to investigate whether UDP-Gal binding in the N-lobe induces conformational changes that affect the CGT active site.\n• Conduct Elastic Network Model (ENM) analyses to assess mechanical coupling and potential allosteric communication between the two lobes.\nMinor Points:\n• Line 376:\nThe sentence \"likely weakening the donor phosphoester bond.....\" is confusing. Typically, binding of Asp437 and Mn²⁺ would stabilize the UDP-Gal and prime it for catalysis, rather than weaken the bond. Please clarify and rephrase accordingly.\n• Figure 4:\nThe figure does not depict key interactions involved in Mn²⁺ coordination. It would strengthen the figure to explicitly show metal coordination with UDP-Gal and relevant catalytic residues.\n• Discussion:\nWhile the manuscript mentions the potential relevance of GLT25D1 in disease, it would benefit from a brief discussion of possible therapeutic opportunities, such as targeting GLT25D1 regulation in collagen-related diseases or cancer.", "reviewer_2": "INTRODUCTION AND OVERALL ASSESSMENT\nThis is a detailed molecular investigation of GLT25D1. The paper includes protein expression, structural analysis by single-particle cryo-EM, and enzymology. From a technical point of view, I personally enjoyed how the careful biochemistry is fully displayed in figure 1 and the elegant application of C3-symmetry expansion to convert the hexamer to a dimer computationally. Overall, the figures are excellent and the text easy to follow. I have some concerns below that need addressing but overall I think this is a great piece of work.\nMAP-MODEL CHECKS\nOverall, the maps and models look good, with the quality of the maps matching what one would expect. However, the one area of ambiguity is the unexplained density between Ala-2 of the substrate and Tyr-400 of the protein in the ternary complex map. It looks like the Tyr could be flipped around to fill it. The authors should confirm that there is no way to do this whilst maintaining clash and stereochemistry values. Figure 5C should be expanded to show density for ligating residues, particularly Tyr-400 and the mystery blob. If local model building can be made to account for that density then that closes the issue, otherwise it should be highlighted and explicitly mentioned in the text.\nMAJOR COMMENTS\nThe issues in the ternary complex map as above.\nHow the peptide was synthesised and purified was not reported. This should go in even if its standard\nThe recent work of Peng et al (Nat Comms, 2024) on the ‘KOGG’ complex made of GLT25D1/ColGalT1 and LH3/PLOD3 should be cited and the relationship between the two works made clear (the focus of the two papers is clearly different).\nThe energy window for the GIF should be reported.\nThe map resolution range in Table in Suppl Table 1 looks weird. I’ve never really known what the Nature family of journals mean with this value in their standard table but I typically read it as the range in the local resolution metric. If so, then the smallest value should match (or be slightly better than) the nominal map resolution above. But assuming that is the case, then it’s not clear what the lower value should be, as the value will depend a lot on input parameters and nominally the lowest resolution feature will be set by box size, which is completely arbitrary. Rest of the stats look good.\nMINOR COMMENTS\n92- Put expression system in main text so its clear to the reader.\n175- in vitro should be in italices\n323 & 328. Not clear why these words are bolded and underlined.\n534- GIF quantum energy filter. Quantum should be capitalised. This makes it sounds like it relies on a quantum effect.\n539- this should be fluence", "reviewer_3": "This manuscript presents cryo-EM structures of human GLT25D1 and its ternary complex with substrates, elucidating the enzyme’s role in collagen galactosylation. While the structural and biochemical data are rigorously obtained and technically sound, major concerns regarding novelty arise due to substantial overlap with two recently published Nature Communications articles (Nat Commun. 2025, 16(1):3624 and Nat Commun. 2025, 16(1):2436).\n1. The Introduction/Discussion must explicitly position this work against these 2025 publications to clarify its unique contributions.\n2. The quality of Figure 1 requires improvement\n-Figure 1A: Abbreviations \"LH/PLODS\" require definition in the legend or main text.\n-Figure 1B: Claims regarding \"dimer\" and \"hexamer\" oligomeric states lack validation. Either add size-exclusion chromatography molecular weight standards to calibrate elution peaks, or remove speculative oligomer labels (\"dimer,\" \"hexamer\") as these states are not biochemically verified.\n-Figure 1C vs. 1D: Content duplication is apparent. Recommend moving Figure 1D to Supplementary Information.\n3. Mutant purification (Lines 216–217): The assertion that failed purification of R61A/V143A/D168A/H235A mutants \"might be the effect of UDP-galactose\" is insufficiently supported. Alternative explanations (e.g., protein misfolding, solubility issues) must be considered or experimentally addressed.\n4. Thermal stability assays (Lines 223–226): Reduced Tm values for mutants only indicate global destabilization, not specifically impaired ligand binding. Direct binding assays (e.g., ITC, SPR) are needed to substantiate claims about UDP-galactose's role in structural stability.\nW135 validation: The rationale for including W135A (not part of initial screening) requires clarification.\n5. No significance testing (e.g., p-values, error bars denoting SD/SEM) is reported for any quantitative data in the whole text (activity assays, thermal shifts, etc.).\n6. Lines 306–308, the importance of Mn²⁺ for enzymatic activity should be contextualized with prior literature citations.\n7. Alternate use of \"hydroxylysine\" and \"hydroxylated lysine\" is confusing.", "reviewer_4": "Relevant Expertise of Reviewer\nI am a biochemistry and structural biologist who works on carbohydrate-processing enzymes, while I can comment on these aspects, I cannot pass comment on the mass spectrometry nor collagen-specific biology as I am not an expert in these.\nOverview\nThe paper details the structure and mechanism of GLT25D1, an important enzyme in the modification of hydroxylysine residues in collagen. Overall, the paper is of high quality with excellent written and visual descriptions of the data. It is a very important piece of work that complements and enhances the recent papers by De Marco et al., (https://doi.org/10.1038/s41467-025-59017-5) and Peng et al., (https://doi.org/10.1038/s41467-025-57768-9). The work is generally robust, and evidence provided is of high quality. It also provides essential new insights including a enzyme UDP/peptide complex and proposed catalytic mechanism. However, I have a few concerns that would warrant consideration and adjustment prior to publication.\nEssential Revisions\n[1] Enzyme Kinetics\nTo assess enzyme kinetics, the authors have chosen to compare the activities of GLT25D1 domains and their mutants relative to the WT enzyme at fixed substrate concentrations. While this approach provides a basic comparison, a more rigorous and informative analysis would ideally to have been for authors to have undertaken full Michaelis-Menten kinetic experiments for all constructs, including the mutants. At a minimum, Michaelis-Menten kinetic experiments should be conducted for the full-length, CGT, and NGT constructs—although the latter may exhibit little to no activity. Given the resources available to the authors, incorporating these experiments should be feasible and would significantly strengthen the study’s conclusions.\nAlso, in relation to the kinetics, it is also unclear how the data has been normalised relative to the WT protein, e.g. in Figure 3 the WT activity against gelatin is 40% of the activity of WT. This is a contradiction, and it is likely authors have normalised activity to WT against Hyl peptide - however this needs clarification. The methods do indicate ‘The final activity was normalized to the GLT25D1 amount quantified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels.’ However, this seems like a very unusual way of normalising activity given protein concentrations have been determined. It is also unclear how the SDS-PAGE normalisation has occurred; did authors quantify band intensities? If so, these values should be provided in the source data files. Still this is a very unusual way to normalise kinetics, and is possibly incorrect, given authors should be using the same quantity of protein in each reaction mix (which they do suggest they are doing within the methods). Please could authors better clarify the mechanism of data collection and normalisation.\nIt is also unclear what the three independent replicates are, and this should be clarified e.g. is this from three entirely separate experimental setups? If so, how many technical replicates in each?\n[2] Bound ion\nIn the NGT domain, authors have modelled a Mn2+ ion, the recent paper by De Marco et al., has indicated by ICP-MS that this is a Ca2+ ion. Please could authors comment on this, the main text suggests this hasn’t been investigated.\nAlso, an important note, the De Marco paper have modelled the galactose differently in their NGT domain, in my opinion the modelling in this paper under review is much more likely to be correct given the highly unlikely puckering and orientation of the galactose in De Marco et al., however this discrepancy should be discussed within the text given its importance. The Peng et al., modelling more closely aligns to the paper under review. However, the UDP in CGT is modelled differently in the Peng et al., paper.\nGiven the large discrepancy, there should be a discussion of the modelling of the UDP-Gal and UDP in this paper against the other papers to resolve the discrepancy - a supplementary figure displaying densities and modelling of the UDP and UDP-Gal from all papers would be particularly useful.\n[3] Cancer Mutants\nLines 340-351, I’m not sure what this passage of text adds to the narrative. The cancer-associated mutations shown in Supp Fig 11 are located across the protein and I think it is ambitious to suggest that mutations within the circled regions all share a mechanistic basis with the other disease mutants. It would have been more useful to have summarised how each cancer-associated mutation could affect structure/activity in table format. I think a more informative narrative is simply stating that mutations in both domains are associated with cancer, indicating the importance of the NGT domain.\n[4] Catalytic mechanism\nLines 284-288. Authors shown convincing evidence for D522 acting as catalytic base in the mechanism, however regarding “Furthermore, the invariant hydroxylysine (Hyl) is stabilized within this cleft by the “YW” motif (Y494 and W495), with its ε-amino group anchored by E523 (Fig. 5B). This configuration positions the 5-hydroxyl group of hydroxylysine in close proximity to the β-phosphate of UDP in CGT active site, appropriate for galactose transfer.” can authors comment on the predicted distance between the proposed nucleophile (Hyl-O) and the predicted position of the anomeric carbon of the galactose, and the suitability of this distance for a concerted Sn2 mechanism. Would authors suggest in slight contradiction to Figure 5F that the D523-Hyl bond is perhaps broken prior to attack? Importantly, the E523 sidechain is not evident in the density map so it is unclear if it is even forming the proposed interaction with the Hyl. While perhaps beyond the scope of the paper, given it is unclear how easily resources can be accessed, MD and QM/MM simulations would have considerably enhanced the manuscript in this regard.\n[5] Terminology\nCryo-EM does not produce ‘electron density’ maps, this term needs updating throughout the text (see: DOI: 10.1002/pro.3060 and DOI: 10.1016/j.sbi.2019.04.006). Also, when density maps are shown in figures the thresholds should be indicated.\n[6] Multimeric state\nGiven the Marco et al., paper proposes that the enzyme is dimeric, there needs to be discussion in the main text acknowledging this work, and a discussion of the discrepancies.\nOther Revisions\n[1] Remove the bold and underlining at the start of sentences e.g. ‘Additionally’, ‘Further Analysis’\n[2] It would be good to see a discussion regarding why NGT is not active from a structural viewpoint.\n[3] Could authors clarify if MS experiments were run once per sample or were there repeats.\nLines 71-74: This part could better describe the motif e.g. presence of Hyp.\nLine 96: It is unclear from the text which of the two elution peaks were used to determine that GLT25D1 was active.\nLine 100: ‘In presence of Mn2+’ needs adding.\nLine122-126: Unclear if the protein used was a mix of the dimer/tetramer SE peak, or an isolated peak.\nLine 172-174: How was significance determined? Also, by eye, it looks like activity of gelatin doesn’t change but does on Hyl containing peptide?\nLine 180-190: Please include surface area for dimer- dimer interactions that comprise the hexamer.\nLine 242 space needed between ‘Fig.’ and ‘1D’\nLine 251: Please specify in main text how H560 and R351 stabilise loop.\nLine 324 “the SVD-associated point mutations L151R and A154P, located in helix α3, play critical roles in stabilizing the domain core fold”\nHere it is inferred the point mutations induce stability, which they don’t, I would rephrase and be more specific about the effects of point mutants, e.g. L151R is introducing a larger, charged residue into a stabilising hydrophobic core.\nLine 336 “Indeed, neither G377R nor R471W significantly”, while this is quite possibly the case, as band intensity has not been calculated and statistical tests not performed, whether the result is ‘significant’ cannot be determined, the sentence needs to be less absolute.\nLine 375, β-pyrophosphate should be β-phosphate\nLine 456-467: Please provide more details regarding how the SDM was performed including primer sequences.\nLine 464: How were cells transferred to BMMY media.\nLine 466: ‘following the manufacturer’s protocol’ it is unclear from the text which protocol this is, as the sentence doesn’t refer to a very specific technique.\nLine 469-470: ‘was applied to Ni-NTA affinity chromatography’ Ni-NTA is the technique, do you mean Ni-NTA resin? Was this gravity flow purification or FPLC-mediated?\nLine 470: ‘After washing’, what was the wash buffer and how much was applied?\nLine 472-472: What ratio of HRV3C to protein did you use? Where was the HRV3C sourced?\nLine 478: How was protein concentration quantified? If by A280 please provide ext.co and Mw used.\nLine 482: How was the hydroxylated peptide synthesised?\nLine 484: I would prefer the concentration of protein to be reported in molarity to enable immediate comparison with the donor and acceptor substrate concentrations.\nLine 526: How were grids glow discharged?\nLine 525-532: It is unclear whether the dimer or tetramer peaks from the SE were used for the unliganded and ternary state. The latter cryo-EM data suggest tetramer was used for the unliganded CTD structure and dimer for ternary structure, if this is not the case, can authors comments on why they see dimer in the latter?\nLine 563: Can the authors check the 275,468 number, a C3-symmetry expansion of 125,162 would suggest 375,486 particles.\nLine 596: ‘Interactive’ not ‘manual’ rebuilding\nLine 802: I think Figure 1A is a little confused as it doesn’t specifically show lysine residues as the modified amino acids. I think a chemical diagram showing each step would have been better but is not essential.\nLine 838: Use red or pink to describe asterisk, not salmon as this is confusing.\nLine 857: Trimer not ‘timer’\nLine 870: The UDP in the diagram is all orange, making the phosphates difficult to ascertain and in contrast to the insert. I would change this to yellow to match insert.\nLine 891: You are missing a few hydrogen bonds in this diagram suggested in main text e.g. K470 to Ala in peptide.\nLine 913: For figure 6 the ‘C’ and ‘D’ are mapped incorrectly in legend, C should be B and D should be C.\nLine 929-930 and 1001-1002: For Sup Figure 1D and E and 7D and E, please provide the corrected, unmasked, masked, and randomised FSC curves.\nLine 970: Trimer not ‘timer’\nLine 980-986: Can you provide uniport identifiers in parentheses after organism’s name in legend.\nLine 1066: Most and least conserved not ‘conservative’.", "reviewer_5": "This manuscript presents a rigorous structural and biochemical dissection of GLT25D1, a collagen galactosyltransferase essential for post-translational modification of hydroxylysine residues. The authors employ high-resolution cryo-EM, structure-guided mutagenesis, and enzymatic assays to map the catalytic site, elucidate substrate specificity, and rationalize the effects of disease-associated mutations. The work is technically sound and clearly presented, and it provides a valuable resource for understanding the molecular mechanism of collagen glycosylation.\nHowever, in considering the broader impact expected for publication in Nature Communications, I am uncertain whether the conceptual advance reaches the necessary threshold. While the structural insights into GLT25D1 are new and welcome, they pertain to a single, well-known enzyme, and the study largely confirms or refines prior expectations (e.g., involvement of the CGT domain in catalysis, recognition of the “X-Hyl-Gly” motif). The physiological or translational implications are discussed but not experimentally explored, and the potential connections to disease, though interesting, remain somewhat speculative.\nMinor comments:\nLine 73: the authors refer to a Gly-X-Y sequence. Y is the symbol for tyrosine, but it seems it is not used to denote tyrosine here\nThere are several instances where \"timer\" has been written in place of \"trimer\" by mistake."}, "author_response": "Point-to-point responses to the reviewers’ comments\nWe greatly thank the insightful and valuable suggestions from reviewers. Our responses\nbelow are in blue.\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nThe manuscript presents a detailed structural and mechanistic study of human\nGLT25D1, an essential collagen O-galactosyltransferase. Using cryo-EM, the authors\nresolve the architecture of GLT25D1, demonstrating a two-lobed structure in which the\nN-lobe binds UDP-Gal non-catalytically, while the C-lobe contains the catalytic CGT\ndomain. Importantly, the study shows that GLT25D1 predominantly forms oligomers, a\nfinding that may have important implications for its structural stability, regulation, or\nbroader cellular function, although dimerization is not essential for enzymatic activity.\nThe authors further integrate biochemical assays and mutational analysis to elucidate\nsubstrate recognition and the effects of disease-related mutations.\nOverall, this study makes a valuable contribution to our understanding of collagen\nglycosylation and opens new mechanistic avenues for further research.\nResponse: We thank the reviewer for the support and the valuable comments. They are\nso helpful to improve our manuscript. We have revised our manuscript accordingly.\nMajor Points:\nThe observation that the non-catalytic N-lobe is critical for enzymatic activity is\nparticularly intriguing and suggests a possible mechanism of allosteric regulation. To\nclarify this aspect:\n• Perform steady-state kinetic analyses (Km and kcat) for both UDP-Gal and peptide\nsubstrates, comparing wild-type GLT25D1 with N-lobe mutants (e.g., D166A).\n• Use molecular dynamics (MD) simulations to investigate whether UDP-Gal binding\nin the N-lobe induces conformational changes that affect the CGT active site.\n• Conduct Elastic Network Model (ENM) analyses to assess mechanical coupling and\npotential allosteric communication between the two lobes.\nResponse: We thank the reviewer for these insightful suggestions. We have revised\naccordingly. The corresponding experimental results have now been added as\nSupplementary Fig. 11, and the related description/discussion has been incorporated\ninto the “Discussion” section of the revised manuscript (Lines 419-445).\nAs recommended, we performed kinetic analyses to compare wild-type GLT25D1\nand the N-lobe mutant D166A. To this end, and given the insufficient yield of the\nsynthesized hydroxylysine-containing acceptor peptide, we used gelatin as the\nsubstrate—a well-established alternative for such assays [1-2]. The kinetic parameters\nobtained for the wild-type (WT) GLT25D1 (Supplementary Fig. S11A-B) were\nconsistent with these studies [1-2]. Notably, compared to the wild-type enzyme, the\nD166A mutant exhibited a ~2.3-fold increase in Km, a ~1.6-fold decrease in kcat, and\na ~3.8-fold reduction in catalytic efficiency (kcat/Km) (Supplementary Fig. S11B),\nsupporting a potential allosteric regulatory mechanism.\nFor further investigation, we agree that MD simulations and ENM analyses can\nprovide valuable insights. Based on our team's expertise, we focused our computational\nefforts on MD simulations. We performed molecular dynamics (MD) simulations of\nGLT25D1 in the apo state and in complex with UDP-galactose bound to the NGT\ndomain (Supplementary Fig. S11C-E). The apo form showed elevated flexibility\nacross the protein, as reflected by broadly increased root-mean-square fluctuation\n(RMSF) values (Supplementary Fig. S11C), indicating structural destabilization in the\nabsence of ligand. Consistently, thermal stability analysis demonstrated that ligand-\nbinding mutations in the NGT domain exhibited a more pronounced destabilizing effect\ncompared to those in the CGT domain (Supplementary Fig. 12). In contrast, the MD\nanalysis showed that UDP-galactose binding at the NGT domain restricted global\nflexibility while selectively increasing RMSF in three localized regions (ΔRMSF <0;\nSupplementary Fig. S11D): region 1 (residues 330–342), which connects the NGT and\nCGT domains; and regions 2 and 3 (residues 375–385 and 387–405, respectively),\nwhich line the substrate-binding site in CGT domain (Supplementary Fig. S11E).\nThese regions are spatially connected: β8 in region 1 runs parallel to β9 immediately\npreceding region 2, and regions 2 and 3 are directly linked. These observations suggest\nthe presence of allosteric communication between the ligand-binding sites in the NGT\nand CGT domains.\nReferences\n[1] Perrin-Tricaud, C., Rutschmann, C. & Hennet, T. Identification of domains and\namino acids essential to the collagen galactosyltransferase activity of GLT25D1. PLoS\nONE 6, e29390 (2011).\n[2] De Marco, M., Rai, S.R., Scietti, L. et al. Molecular structure and enzymatic\nmechanism of the human collagen hydroxylysine galactosyltransferase\nGLT25D1/COLGALT1. Nat Commun 16, 3624 (2025).\nSupplementary Figure 11. Enzyme kinetics and molecular dynamics analysis of\nGLT25D1.\nA, Enzyme kinetics of wild-type (WT) GLT25D1 and its variants, including the NGT\nmutant D166A, the isolated CGT domain, and the isolated NGT domain. Data are\npresented as mean ± SD from three independent replicates. Please refer to Methods\nsection for experimental details.\nB, Enzyme kinetical parameters, corresponding to the analysis in (A).\nC, RMSF (root-mean-square fluctuation) curves from molecular dynamics simulations\nof apo-GLT25D1 (blue curve) and liganded GLT25D1 (with UDP-Galactose bound to\nNGT domain) (orange curve).\nD, Difference in RMSF (ΔRMSF) curve (red) between apo-GLT25D1 and liganded\nGLT25D1 (with UDP-galactose bound to NGT domain). Three localized regions\n(regions 1-3) exhibiting increased RMSF upon ligand binding to the NGT domain are\nmarked with shaded boxes in (C) and (D).\nE, Three regions (regions 1-3) identified in (C) and (D) are mapped to the ternary\ncomplex of GLT25D1, which contains UDP-galactose in NGT domain, UDP and a\nhydroxylysine-containing acceptor peptide in CGT domain. Specifically, region 1\n(residues 330–342) connects the NGT and CGT domains; regions 2 and 3 (residues\n375–385 and 387–405, respectively) line the substrate-binding site in CGT domain.\nThese three regions are spatially connected: β8 in region 1 runs parallel to β9\nimmediately preceding region 2, and regions 2 and 3 are directly linked.\nMinor Points:\n• Line 376:\nThe sentence \"likely weakening the donor phosphoester bond.....\" is confusing.\nTypically, binding of Asp437 and Mn²⁺ would stabilize the UDP-Gal and prime it for\ncatalysis, rather than weaken the bond. Please clarify and rephrase accordingly.\nResponse: We thank the reviewer for this suggestion. We have now clarified and\nrephrased the confusing sentence in the manuscript (Lines 383-386 in the “Discussions”\nsection) as follows:\n“A manganese (II) ion, coordinated by residue D437 of “EDD” motif (Glu435, Asp436\nand Asp437), stabilize the donor diphosphate and primes it for catalysis.”\n• Figure 4:\nThe figure does not depict key interactions involved in Mn²⁺ coordination. It would\nstrengthen the figure to explicitly show metal coordination with UDP-Gal and relevant\ncatalytic residues.\nResponse: We thank the reviewer for this suggestion. In response, we have revised Fig.\n4D to explicitly illustrate metal coordination with donor diphosphate and residue D437.\nAs suggested, we have also shown D522 in Fig. 4D, which is identified as the key\ncatalytic base in our later analysis.\n• Discussion:\nWhile the manuscript mentions the potential relevance of GLT25D1 in disease, it would\nbenefit from a brief discussion of possible therapeutic opportunities, such as targeting\nGLT25D1 regulation in collagen-related diseases or cancer.\nResponse: We thank for this insightful suggestion. In response, we have added a brief\ndiscussion of possible therapeutic opportunities in the “Discussion” section (Lines 464-\n475). The added text now reads as follows:\n“The molecular characterization of disease-linked GLT25D1 mutations improves our\nunderstanding of pathogenic mechanism and provides a foundation for developing\nprecise biochemical diagnosis to probe GLT25D1 dysregulation in collagen-related\ndiseases. The distinct structural and functional deficiencies caused by specific variants\nmay correlate with varying clinical severities and subtypes, awaiting further\ninvestigation. From a therapeutic perspective, these mechanistic insights suggest that\nrestoring enzymatic activity—either through stabilization of misfolded proteins, or\ngene-based approaches—could represent viable treatment strategies. Moreover, the\nassociation of GLT25D1 dysfunction with both inherited vascular/musculoskeletal\ndisorders and tumors highlights the potential of targeting GLT25D1 in both rare\ndiseases and oncology.”\nReviewer #2 (Remarks to the Author):\nINTRODUCTION AND OVERALL ASSESSMENT\nThis is a detailed molecular investigation of GLT25D1. The paper includes protein\nexpression, structural analysis by single-particle cryo-EM, and enzymology. From a\ntechnical point of view, I personally enjoyed how the careful biochemistry is fully\ndisplayed in figure 1 and the elegant application of C3-symmetry expansion to convert\nthe hexamer to a dimer computationally. Overall, the figures are excellent and the text\neasy to follow. I have some concerns below that need addressing but overall I think this\nis a great piece of work.\nResponse: We greatly thank for the professional evaluation and support of our work.\nWe have made the revisions accordingly.\nMAP-MODEL CHECKS\nOverall, the maps and models look good, with the quality of the maps matching what\none would expect. However, the one area of ambiguity is the unexplained density between\nAla-2 of the substrate and Tyr-400 of the protein in the ternary complex map.\nIt looks like the Tyr could be flipped around to fill it. The authors should confirm that\nthere is no way to do this whilst maintaining clash and stereochemistry values. Figure\n5C should be expanded to show density for ligating residues, particularly Tyr-400 and\nthe mystery blob. If local model building can be made to account for that density then\nthat closes the issue, otherwise it should be highlighted and explicitly mentioned in the\ntext.\nResponse: We thank the reviewer for this valuable suggestion. Following reviewer’s\nrecommendation, we explored flipping Tyr-400 and found that the resulting\nconformation indeed fits well into the unexplained density (Response Figure 1).\nAccordingly, we have updated the structural model with flipped Tyr-400 in the PDB\ndatabase and expanded Figure 5C to clearly display the density for the acceptor peptide\nand its surrounding residues, including Tyr-400.\nResponse Figure 1. The acceptor peptide (yellow) and its surrounding GLT25D1\nresidues (blue) are superimposed with their corresponding densities (grey mesh).\nThe arrows indicate Tyr-400 (Y400) before and after side chain flipping.\nMAJOR COMMENTS\nThe issues in the ternary complex map as above.\nResponse: As described above, we have now addressed this issue as suggested by the\nreviewer.\nHow the peptide was synthesized and purified was not reported. This should go in even\nif its standard.\nResponse: We thank the reviewer for raising this question. We have now included the\nsource of the hydroxylysine-containing peptide and a corresponding synthesis reference\nin the “Methods” section (Lines 571-575).\nThe recent work of Peng et al (Nat Comms, 2024) on the ‘KOGG’ complex made of\nGLT25D1/ColGalT1 and LH3/PLOD3 should be cited and the relationship between the\ntwo works made clear (the focus of the two papers is clearly different).\nResponse: We thank the reviewer for this insightful suggestion. This point is also\nrelated to Reviewer #3 point #1. We have revised accordingly, positioning our work in\nthe context of recent findings and clarifying our unique contributions (Lines 476-497\nin “Discussions” section) as briefly mentioned below:\nDuring the preparation or review of our manuscript, two related studies on\nGLT25D1 were published [1-2]:\n[1]. Peng et al., Nat Commun. 2025, 16(1):2436, published during our manuscript\npreparation;\n[2]. De Marco et al., Nat Commun. 2025, 16(1):3624, published during the review of\nour manuscript.\nSpecifically, Peng et al. described cryo-EM structures of the GLT25D1–LH3\ncomplex bound to UDP or UDP-galactose, while De Marco et al. used crystallography\nmethod to determine the structures of GLT25D1 bound to UDP or UDP-galactose. As\nalso noted by this reviewer and reviewer #4, three studies—though differing in\nmethodological emphasis (cryo-EM vs. crystallography) and research focus\n(GLT25D1-LH3 complex vs. GLT25D1 alone)—complement and enhance one another.\nIn particular, all three works independently reported the dimeric architecture of\nGLT25D1 and the unexpected binding of UDP-galactose in the NGT domain.\nBeyond these consensus findings, our study provides several unique contributions.\nMost importantly, we uniquely present GLT25D1 in a ternary complex with UDP/UDP-\ngalactose and a designed hydroxylysine-containing acceptor substrate, supported by\ndetailed structure-based functional characterization. Such a ternary complex is essential\nfor a deeper understanding of a protein-targeting glycosyltransferase like GLT25D1,\nyet was not available in the other two studies. This approach has enabled us to uniquely\ndefine the precise catalytic mechanism and elucidate the molecular determinants\ngoverning collagen recognition. Furthermore, in addition to the dimeric form\nconsistently reported across all three studies, we have identified a hexameric assembly\nof GLT25D1, an architectural feature not previously described in this enzyme family.\nWe also performed a detailed functional analysis of disease-related mutations,\nclarifying their mechanistic impact on GLT25D1 dysfunction—an aspect not explored\nin the other studies.\nReferences\n[1] Peng, J., Li, W., Yao, D. et al. The structural basis for the human procollagen lysine\nhydroxylation and dual-glycosylation. Nat Commun 16, 2436 (2025).\n[2] De Marco, M., Rai, S.R., Scietti, L. et al. Molecular structure and enzymatic\nmechanism of the human collagen hydroxylysine galactosyltransferase\nGLT25D1/COLGALT1. Nat Commun 16, 3624 (2025).\nThe energy window for the GIF should be reported.\nResponse: We thank the reviewer for this comment. The energy window used for the\nGIF (20ev) has now been specified in the Methods section (Line 641).\nThe map resolution range in Table in Suppl Table 1 looks weird. I’ve never really known\nwhat the Nature family of journals mean with this value in their standard table but I\ntypically read it as the range in the local resolution metric. If so, then the smallest value\nshould match (or be slightly better than) the nominal map resolution above. But\nassuming that is the case, then it’s not clear what the lower value should be, as the\nvalue will depend a lot on input parameters and nominally the lowest resolution feature\nwill be set by box size, which is completely arbitrary. Rest of the stats look good.\nResponse: We thank the reviewer for this professional comment. We agree that the\nreported resolution range can be influenced by multiple parameters and its\ninterpretation may vary. We also note that some publications have omitted this value.\nAccordingly, we have removed the resolution range entry from Supplementary Table\n1 in the revised manuscript. The rest of the table remains unchanged.\nMINOR COMMENTS\n92- Put expression system in main text so its clear to the reader.\nResponse: We thank the reviewer for this suggestion. In response, we have now\nincluded this information in the main text (Lines 96–97).\n175- in vitro should be in italices\nResponse: We have corrected this and have italicized “in vitro” throughout the revised\nmanuscript.\n323 & 328. Not clear why these words are bolded and underlined.\nResponse: We apology for the confusion. The bold and underlined formatting was\noriginally used to indicate conceptual hierarchy during the drafting stage. We have now\nreviewed the entire manuscript and removed all such formatting.\n534- GIF quantum energy filter. Quantum should be capitalised. This makes it sounds\nlike it relies on a quantum effect.\nResponse: We apology for this confusion. We have corrected this.\n539- this should be fluence\nResponse: We have corrected this.\nReviewer #3 (Remarks to the Author):\nThis manuscript presents cryo-EM structures of human GLT25D1 and its ternary\ncomplex with substrates, elucidating the enzyme’s role in collagen galactosylation.\nWhile the structural and biochemical data are rigorously obtained and technically\nsound, major concerns regarding novelty arise due to substantial overlap with two\nrecently published Nature Communications articles (Nat Commun. 2025, 16(1):3624\nand Nat Commun. 2025, 16(1):2436).\n1. The Introduction/Discussion must explicitly position this work against these 2025\npublications to clarify its unique contributions.\nResponse: We thank the reviewer for acknowledging the technical soundness of our\nwork. As recommended, we have revised the manuscript to explicitly compare our\nstudy with the recently published works and to clarify our unique contributions (see\nLines 476-497 in the “Discussion” section) as briefly mentioned below:\nDuring the preparation or review of our manuscript, two related studies on\nGLT25D1 were published [1-2]:\n[1]. Peng et al., Nat Commun. 2025, 16(1):2436, published during our manuscript\npreparation;\n[2]. De Marco et al., Nat Commun. 2025, 16(1):3624, published during the review of\nour manuscript.\nSpecifically, Peng et al. described cryo-EM structures of the GLT25D1–LH3\ncomplex bound to UDP or UDP-galactose, while De Marco et al. used crystallography\nmethod to determine the structures of GLT25D1 bound to UDP or UDP-galactose. As\nalso noted by Reviewers #1 and #4, these three studies—though differing in\nmethodological emphasis (cryo-EM vs. crystallography) and research focus\n(GLT25D1-LH3 complex vs. GLT25D1 alone)—complement and enhance one another.\nIn particular, all three works independently reported the dimeric architecture of\nGLT25D1 and the unexpected binding of UDP-galactose in the NGT domain.\nBeyond these consensus findings, our study provides several unique contributions.\nMost importantly, we uniquely present GLT25D1 in a ternary complex with UDP/UDP-\ngalactose and a designed hydroxylysine-containing acceptor substrate, supported by\ndetailed structure-based functional characterization. Such a ternary complex is essential\nfor a deeper understanding of a protein-targeting glycosyltransferase like GLT25D1,\nyet was not available in the other two studies. This approach has enabled us to uniquely\ndefine the precise catalytic mechanism and elucidate the molecular determinants\ngoverning collagen recognition. Furthermore, in addition to the dimeric form\nconsistently reported across all three studies, we have identified a hexameric assembly\nof GLT25D1, an architectural feature not previously described in this enzyme family.\nWe also performed a detailed functional analysis of disease-related mutations,\nclarifying their mechanistic impact on GLT25D1 dysfunction—an aspect not explored\nin the other studies.\nReferences\n[1] Peng, J., Li, W., Yao, D. et al. The structural basis for the human procollagen lysine\nhydroxylation and dual-glycosylation. Nat Commun 16, 2436 (2025).\n[2] De Marco, M., Rai, S.R., Scietti, L. et al. Molecular structure and enzymatic\nmechanism of the human collagen hydroxylysine galactosyltransferase\nGLT25D1/COLGALT1. Nat Commun 16, 3624 (2025).\n2. The quality of Figure 1 requires improvement\n-Figure 1A: Abbreviations \"LH/PLODS\" require definition in the legend or main text.\n-Figure 1B: Claims regarding \"dimer\" and \"hexamer\" oligomeric states lack validation.\nEither add size-exclusion chromatography molecular weight standards to calibrate\nelution peaks, or remove speculative oligomer labels (\"dimer,\" \"hexamer\") as these\nstates are not biochemically verified.\n-Figure 1C vs. 1D: Content duplication is apparent. Recommend moving Figure 1D to\nSupplementary Information.\nResponse: We thank the reviewer for these valuable suggestions. In response, the\nabbreviations “LH/PLODs” have now been clearly defined in the revised legend of\nFigure 1A. The oligomer labels in Figure 1B have been removed. Regarding Figure 1C\nand 1D, we have carefully considered the reviewer’s suggestion and would like to\nexplain our rationale for this organization. Figure 1C and 1D represent two independent\nassay methods, enabling cross-validation of the findings. Moreover, the use of our\ndesigned pure peptide in Figure 1D–F allows direct mass spectrometry-based detection\nof modifications, which is essential for confirming GLT25D1-mediated galactosylation\n(Fig. 1D), illustrating donor specificity (Fig. 1D vs. 1E), and demonstrating the\nenzyme’s inability to elongate galactosylated substrates (Fig. 1D vs. 1F). Therefore,\npresenting these results together would provide more comprehensive insights into the\nfunction of GLT25D1, which was also mentioned by Reviewer #2. We wish the\nreviewer finds this rationale acceptable, but we remain open to further discussion and\nadjustments should the reviewer still have concerns or suggestions regarding the figure\norganization.\n3. Mutant purification (Lines 216–217): The assertion that failed purification of\nR61A/V143A/D168A/H235A mutants \"might be the effect of UDP-galactose\" is\ninsufficiently supported. Alternative explanations (e.g., protein misfolding, solubility\nissues) must be considered or experimentally addressed.\nResponse: We thank the reviewer for this insightful suggestion. Following the\ncomment, we have revised the statement to incorporate alternative explanations. It now\nreads: “a result that may be influenced by the stabilizing effect of UDP-galactose,\nthough other factors such as protein misfolding could also contribute.” In addition, to\nfurther tone down this claim, we have relocated the statement to the “Discussion”\nsection (Lines 416-417).\n4. Thermal stability assays (Lines 223–226): Reduced Tm values for mutants only\nindicate global destabilization, not specifically impaired ligand binding. Direct binding\nassays (e.g., ITC, SPR) are needed to substantiate claims about UDP-galactose's role\nin structural stability.\nW135 validation: The rationale for including W135A (not part of initial screening)\nrequires clarification.\nResponse: We thank the reviewer for this insightful comment and we fully agree with\nthe reviewer. In response, we have significantly toned down our claim and have moved\nall relevant interpretations from “Results” to “Discussion” section (Lines 432-435).\nThere, we present this as a possible interpretation, integrated with the new molecular\ndynamics (MD) simulation data performed at the suggestion of Reviewer #1. Below is\nour explanation for these changes:\nWe initially designed this thermal stability assays to investigate the potential\nregulatory function of non-catalytic NGT domain. As the reviewer rightly noted, direct\nbinding assays can substantiate this claim. However, since the GLT25D1 is purified\nwith endogenous UDP-galactose tightly bound to its NGT domain, we could not obtain\nligand-free protein for binding analysis. As reviewer #1 pointed out (point #1), the\neffects of NGT mutants suggest a possible mechanism of allosteric regulation.\nFollowing this suggestion, we have performed molecular dynamics (MD) simulations\nof GLT25D1 in the apo state and in complex with UDP-galactose bound to the NGT\ndomain (Supplementary Fig. S11C-E). From the MD results, the apo form showed\nelevated flexibility across the protein, as reflected by broadly increased root-mean-\nsquare fluctuation (RMSF) values (Supplementary Fig. S11C), indicating structural\ndestabilization in the absence of ligand. This aligns with our experimental observation\nthat ligand-binding mutations in the NGT domain generally cause more pronounced\ndestabilization than those in the CGT domain (Supplementary Fig. 12).\nRegarding the W135A mutant, we apologize for the oversight in not clearly stating\nits rationale in the initial submission. W135 stacks against the galactose moiety of the\ndonor substrate, as shown in Fig. 3B, and was indeed included in our initial mutant\nscreening. We have now corrected this omission in the revised manuscript (Line 224).\n5. No significance testing (e.g., p-values, error bars denoting SD/SEM) is reported for\nany quantitative data in the whole text (activity assays, thermal shifts, etc.).\nResponse: We thank the reviewer for this valuable suggestion. In response, we have\nnow incorporated p-values into all relevant activity assay figures. For clarity, error bars\nrepresenting SD were already included in the original submission, and we have\nreconfirmed their presence during revision. Additionally, as suggested, we have now\nalso provided both p-values and error bars for the thermal shift data (Supplementary\nFig. 12).\n6. Lines 306–308, the importance of Mn²⁺ for enzymatic activity should be\ncontextualized with prior literature citations.\nResponse: We thank the reviewer for the suggestion. We have cited relevant literatures\n[1-3] (Line 314 in the revised text) to emphasize the importance of Mn²⁺ for enzymatic\nactivity in revised manuscript.\nReferences\n[1] Spiro, M.J. & Spiro, R.G. Studies on the biosynthesis of the hydroxylsine-linked\ndisaccharide unit of basement membranes and collagens. II. Kidney\ngalactosyltransferase. The Journal of biological chemistry 246, 4910-4918 (1971).\n[2] Myllylä, R., Risteli, L. & Kivirikko, K.I. Assay of collagen-galactosyltransferase\nand collagen-glucosyltransferase activities and preliminary characterization of enzymic\nreactions with transferases from chick-embryo cartilage. European journal of\nbiochemistry 52, 401-410 (1975).\n[3] Kivirikko, K.I. & Myllylä, R. Collagen glycosyltransferases. International review\nof connective tissue research 8, 23-72 (1979)\n7. Alternate use of \"hydroxylysine\" and \"hydroxylated lysine\" is confusing.\nResponse: We thank the reviewer for this helpful suggestion. We have revised to\nconsistently use the term \"hydroxylysine\" instead of “hydroxylated lysine” throughout\nthe manuscript.\nReviewer #4 (Remarks to the Author):\nRelevant Expertise of Reviewer\nI am a biochemistry and structural biologist who works on carbohydrate-processing\nenzymes, while I can comment on these aspects, I cannot pass comment on the mass\nspectrometry nor collagen-specific biology as I am not an expert in these.\nOverview\nThe paper details the structure and mechanism of GLT25D1, an important enzyme in\nthe modification of hydroxylysine residues in collagen. Overall, the paper is of high\nquality with excellent written and visual descriptions of the data. It is a very important\npiece of work that complements and enhances the recent papers by De Marco et al.,\n(https://doi.org/10.1038/s41467-025-59017-5) and Peng et al.,\n(https://doi.org/10.1038/ s41467-025-57768-9). The work is generally robust, and\nevidence provided is of high quality. It also provides essential new insights including a\nenzyme UDP/peptide complex and proposed catalytic mechanism. However, I have a\nfew concerns that would warrant consideration and adjustment prior to publication.\nResponse: We thank the reviewer for the support and the important comments to\nimprove the manuscript. We have revised our manuscript accordingly.\n[1] Enzyme Kinetics\nTo assess enzyme kinetics, the authors have chosen to compare the activities of\nGLT25D1 domains and their mutants relative to the WT enzyme at fixed substrate\nconcentrations. While this approach provides a basic comparison, a more rigorous and\ninformative analysis would ideally to have been for authors to have undertaken full\nMichaelis-Menten kinetic experiments for all constructs, including the mutants. At a\nminimum, Michaelis-Menten kinetic experiments should be conducted for the full-\nlength, CGT, and NGT constructs—although the latter may exhibit little to no activity.\nGiven the resources available to the authors, incorporating these experiments should\nbe feasible and would significantly strengthen the study’s conclusions.\nResponse: We thank for this insightful comment. We fully agree that kinetic analysis\ncan strengthen our study. As recommended, we have performed kinetic experiments for\nthe full length, the isolated CGT domain, and NGT domain (Supplementary Fig. 11A).\nThe kinetic parameters obtained for the wild-type (WT) GLT25D1 (Supplementary Fig.\nS11B) were consistent with other studies [1-2]. Moreover, the NGT domain indeed\nshowed negligible activity, as anticipated.\nAdditionally, following a related suggestion from Reviewer #1 (point #1), we have\nextended our kinetic analysis to include the NGT mutant D166A and conducted\nmolecular dynamics (MD) simulations to explore the potential mechanism of allosteric\nregulation. These new kinetic and computational data are now included as\nSupplementary Figure 11 and collectively provide deeper insights into the function of\nGLT25D1 (please see related contents in “Discussion” section Lines 419-448).\nReferences\n[1] Perrin-Tricaud, C., Rutschmann, C. & Hennet, T. Identification of domains and\namino acids essential to the collagen galactosyltransferase activity of GLT25D1. PLoS\nONE 6, e29390 (2011).\n[2] De Marco, M., Rai, S.R., Scietti, L. et al. Molecular structure and enzymatic\nmechanism of the human collagen hydroxylysine galactosyltransferase\nGLT25D1/COLGALT1. Nat Commun 16, 3624 (2025).\nAlso, in relation to the kinetics, it is also unclear how the data has been normalised\nrelative to the WT protein, e.g. in Figure 3 the WT activity against gelatin is 40% of the\nactivity of WT. This is a contradiction, and it is likely authors have normalised activity\nto WT against Hyl peptide - however this needs clarification. The methods do indicate\n‘The final activity was normalized to the GLT25D1 amount quantified by sodium\ndodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels.’ However, this\nseems like a very unusual way of normalising activity given protein concentrations have\nbeen determined. It is also unclear how the SDSPAGE normalisation has occurred; did\nauthors quantify band intensities? If so, these values should be provided in the source\ndata files. Still this is a very unusual way to normalise kinetics, and is possibly incorrect,\ngiven authors should be using the same quantity of protein in each reaction mix (which\nthey do suggest they are doing within the methods). Please could authors better clarify\nthe mechanism of data collection and normalisation.\nResponse: We thank the reviewer for raising this important point. The reviewer is\ncorrect in noting the wild-type (WT) activity against gelatin is normalized to WT\nactivity against the hydroxylysine peptide. We have now clarified this in the Methods\nsection (Lines 592-594 in “Methods” section).\nRegarding the use of SDS-PAGE for quantification, this was implemented because\ninitial purity assessments (Supplementary Fig. 5) revealed impurities for certain\nmutants, and we also observed some inaccuracy in protein concentration determinations\nof certain mutants. To ensure accurate activity comparisons across variants, we\ntherefore used SDS-PAGE band intensity quantification to normalize the enzyme\namounts for WT GLT25D1 and its mutants, rather than relying solely on pre-assay\nconcentration measurements. As helpfully suggested, we have clarified this in the\n“Methods” section (Lines 588-590) and provided the original SDS-PAGE data in the\nsource data file.\nIt is also unclear what the three independent replicates are, and this should be clarified\ne.g. is this from three entirely separate experimental setups? If so, how many technical\nreplicates in each?\nResponse: We apologize for the confusion. We have now clarified the experimental\ndetails. For each enzyme or variant, we independently prepared three separate\nenzymatic reactions. Each reaction was then individually measured using the UDP-\nGlo™ Glycosyltransferase Assay kit (Promega).\n[2] Bound ion\nIn the NGT domain, authors have modelled a Mn2+ ion, the recent paper by De Marco\net al., has indicated by ICP-MS that this is a Ca2+ ion. Please could authors comment\non this, the main text suggests this hasn’t been investigated.\nAlso, an important note, the De Marco paper have modelled the galactose differently\nin their NGT domain, in my opinion the modelling in this paper under review is much\nmore likely to be correct given the highly unlikely puckering and orientation of the\ngalactose in De Marco et al., however this discrepancy should be discussed within the\ntext given its importance. The Peng et al.,modelling more closely aligns to the paper\nunder review. However, the UDP in CGT is modelled differently in the Peng et al., paper.\nGiven the large discrepancy, there should be a discussion of the modelling of the UDP-\nGal and UDP in this paper against the other papers to resolve the discrepancy - a\nsupplementary figure displaying densities and modelling of the UDP and UDP-Gal\nfrom all papers would be particularly useful.\nResponse: We thank the reviewer for these valuable observations and suggestions. We\nhave accordingly incorporated a detailed discussion about the discrepancies in metal\nion identity and substrate modeling among these independently determined structures\n(Lines 497-515 in the “Discussions” section) as briefly mentioned below:\nRegarding the metal ion, our cryo-EM sample for the ternary complex was\nprepared in the presence of 1 mM MnCl , and accordingly, we modelled Mn²⁺ ions in\n2\nthe ligand binding sites of both the NGT and CGT domains. De Marco et al. reported a\nCa²⁺ ion at the equivalent site of the NGT domain and a Mn2+ ion in the CGT domain.\nIt is possible that metal ion occupancy may be influenced by cellular or experimental\nconditions, and further investigation will be needed to determine the physiologically\nrelevant cofactor.\nRegarding ligand modeling, there are also variations as mentioned by the reviewer.\nAs suggested, we have added a new Supplementary Figure 13 that directly compares\nthe cryo-EM densities and ligand models across these studies. This provides a clear\nvisual reference to help readers evaluate the different structural interpretations:\n(1) In the NGT domain, the galactose moiety of UDP-galactose adopts a favorable\nconformation in our structure, consistent with that reported by Peng et al., but distinct\nfrom the conformation reported by De Marco et al. (Supplementary Fig. 13A-C). The\nUDP moiety, however, is consistent between our model and that of De Marco et al.,\nwhereas Peng et al. modeled the uracil group in a flipped orientation (Supplementary\nFig. 13A-C).\n(2) In the CGT domain, the UDP orientation is consistent between our study and De\nMarco et al., but again differs from the flipped uracil conformation reported by Peng et\nal. (Supplementary Fig. 13A-C).\nSupplementary Figure 13. Comparison of nucleotide-ligand modeling and\ncorresponding cryo-EM densities among three recent independent structural\nstudies on GLT25D1.\nA, GLT25D1 ternary complex with UDP/UDP-Galactose and a hydroxylysine-\ncontaining acceptor peptide (this study).\nB, GLT25D1/LH3 bound to UDP/UDP-galactose (Peng et al. Nat Commun, 16, 2436\n(2025)).\nC, GLT25D1 bound to UDP/UDP-Galactose (De Marco et al. Nat Commun, 16: 3624-\n3624 (2025)).\nFor each panel, the top section depicts the UDP-galactose bound to the NGT domain,\nand the bottom section shows the UDP bound to the CGT domain. Arrows highlight\nkey modeling discrepancies among the corresponding ligands.\n[3] Cancer Mutants\nLines 340-351, I’m not sure what this passage of text adds to the narrative. The cancer-\nassociated mutations shown in Supp Fig 11 are located across the protein and I think it\nis ambitious to suggest that mutations within the circled regions all share a mechanistic\nbasis with the other disease mutants. It would have been more useful to have\nsummarised how each cancer-associated mutation could affect structure/activity in\ntable format. I think a more informative narrative is simply stating that mutations in\nboth domains are associated with cancer, indicating the importance of the NGT domain.\nResponse: We thank the reviewer for this insightful suggestion. We fully agree and\nhave revised the text accordingly (Lines 352-357 in the section “Molecular insights into\npathogenic mutations of GLT25D1”). As suggested, we now emphasize that cancer-\nassociated mutations are distributed across both domains, and to strengthen this point,\nwe specifically highlight four cancer-associated mutations, which locate to key ligand-\nbinding interfaces in both domains: R147H, R147C, and D168H in the NGT domain,\nand G377D in the CGT domain. The revised text now reads:\n“These mutations are distributed across both the NGT and CGT domains\n(Supplementary Fig. 10), consistent with the functional importance of each domain.\nNotably, four cancer-associated mutations (R147H, R147C, D168H, and G377D) are\nsituated at key ligand-binding interfaces and are likely to impair GLT25D1 function:\nR147H, R147C, and D168H in the NGT domain (Fig. 3B), and G377D in the CGT\ndomain (Fig. 4D).”\n[4] Catalytic mechanism\nLines 284-288. Authors shown convincing evidence for D522 acting as catalytic base\nin the mechanism, however regarding “Furthermore, the invariant hydroxylysine (Hyl)\nis stabilized within this cleft by the “YW” motif (Y494 and W495), with its ε-\namino group anchored by E523 (Fig. 5B). This configuration positions the 5-hydroxyl group\nof hydroxylysine in close proximity to the β-phosphate of UDP in CGT active site,\nappropriate for galactose transfer.” can authors comment on the predicted distance\nbetween the proposed nucleophile (Hyl-O) and the predicted position of the anomeric\ncarbon of the galactose, and the suitability of this distance for a concerted Sn2\nmechanism. Would authors suggest in slight contradiction to Figure 5F that the\nD523-Hyl bond is perhaps broken prior to attack? Importantly, the E523 sidechain is not\nevident in the density map so it is unclear if it is even forming the proposed interaction\nwith the Hyl. While perhaps beyond the scope of the paper, given it is unclear how easily\nresources can be accessed, MD and QM/MM simulations would have considerably\nenhanced the manuscript in this regard.\nResponse: We thank the reviewer for these insightful comments and constructive\nsuggestions regarding the catalytic mechanism.\nTo address the question of the reaction distance and mechanism, we performed a\nDALI search using the CTD domain of GLT25D1, which identified a structural\nhomolog: the bacterial galactosyltransferase Aaβ4GalT complexed with UDP-galactose\n(PDB ID: 8XOC). Although the sequence identity is low (<20%), GLT25D1 CTD\ndomain and Aaβ4GalT can be superimposed with an RMSD of 3.2 Å over 171 aligned\nresidues (Response Fig. 2A), revealing a well-conserved architecture of their active\nsites (Response Fig. 2B). Based on this structural superposition, we generated a\ncomposite model of the GLT25D1 CGT domain with UDP-galactose (Response Fig.\n2B). In this composite model, the nucleophile (Hyl-O) is positioned for an in-line attack\non the anomeric carbon (C1) of the galactose moiety, with modeled distance of 4.9 Å\n(Response Fig. 2B). While we acknowledge that this value is a homology-based\nestimate and local conformational dynamics may occur during catalysis process in\nreality, the overall geometry appears to support the catalytic mechanism proposed in\nFig. 5F.\nRegarding the mechanism illustrated in Fig. 5F, we apologize for confusion due to\nthe lack of explanation. The figure legend has been revised to clarify the proposed\ncatalytic step as follows: “Specifically, D522 deprotonates the 5-hydroxyl group of\nhydroxylysine, activating it as a nucleophile to attack the anomeric carbon (C1) of the\ngalactose moiety.” As for E523, although our structural and biochemical evidence\nsuggest its potential role in interacting with the hydroxylysine side chain, we\nacknowledge that the corresponding electron density is not clearly resolved. Therefore,\nwe have revised the main text to clearly state that E523 is “potentially” involved in this\ninteraction (Line 292).\nWe fully agree with the reviewer that molecular dynamics (MD) and QM/MM\nsimulations would provide deeper mechanistic insights. While such comprehensive\ncomputational studies lie beyond the scope of the current structural and biochemical\nwork, we believe the structural framework and mechanistic hypotheses established here\nwill serve as a solid foundation for future in-depth investigations.\nResponse Figure 2. Structural comparison between GLT25D1 CGT domain (with\nUDP in CGT domain) and bacterial galactosyltransferase Aaβ4GalT (complexed\nwith UDP-galactose).\nA, Structural alignment between GLT25D1 CGT domain (this study; in blue; with UDP\nand Hyl shown as sticks) and Aaβ4GalT (PDB ID: 8XOC; in green; with UDP-\ngalactose shown as sticks).\nB, Close-up view of the ligand binding sites between GLT25D1 CGT domain and\nAaβ4GalT, as marked by the dashed box in (A). In this estimated composite model, the\nnucleophile (Hyl-O) is positioned for an in-line attack on the anomeric carbon (C1) of\nthe galactose moiety, with modeled distance of 4.9 Å.\n[5] Terminology\nCryo-EM does not produce ‘electron density’ maps, this term needs updating\nthroughout the text (see: DOI: 10.1002/pro.3060 and DOI: 10.1016/j.sbi.2019.04.006).\nAlso, when density maps are shown in figures the thresholds should be indicated.\nResponse: We thank the reviewer for this correction and for providing the relevant\nreferences. We fully agree and have therefore replaced all instances of \"electron density\nmap\" with \"EM map\" throughout the manuscript. Furthermore, as suggested, we have\nnow specified the display thresholds for all density maps in their respective figure\nlegends.\n[6] Multimeric state\nGiven the Marco et al., paper proposes that the enzyme is dimeric, there needs to be\ndiscussion in the main text acknowledging this work, and a discussion of the\ndiscrepancies.\nResponse: We thank the reviewer for this constructive suggestion. We have revised the\nmanuscript accordingly to acknowledge recent related works and to discuss\ndiscrepancies regarding the oligomeric assembly of GLT25D1 (Line 492-495 in\n“Discussion” section). While all three independent studies—including ours, De Marco\net al.[1], and Peng et al.[2]—consistently report the dimeric form of GLT25D1, our\nstudy further identifies a hexameric assembly, an architectural feature not previously\ndescribed in this enzyme family. These findings have now been incorporated into the\n\"Discussion\" section (Lines 476-497), where we position our work in the context of\nrecent structural studies and clarify the unique contributions of our findings.\nReferences\n[1] De Marco, M., Rai, S.R., Scietti, L. et al. Molecular structure and enzymatic\nmechanism of the human collagen hydroxylysine galactosyltransferase\nGLT25D1/COLGALT1. Nat Commun 16, 3624 (2025).\n[2] Peng, J., Li, W., Yao, D. et al. The structural basis for the human procollagen lysine\nhydroxylation and dual-glycosylation. Nat Commun 16, 2436 (2025).\nOther Revisions\n[1] Remove the bold and underlining at the start of sentences e.g. ‘Additionally’,\n‘Further Analysis’\nResponse: We have removed all the bold and underlining labels throughout the\nmanuscript.\n[2] It would be good to see a discussion regarding why NGT is not active from a\nstructural viewpoint.\nResponse: We thank for this suggestion. In response, we have added a discussion about\nthe reason why NGT is not active (Lines 402-410 in “Discussions” section). It reads as\nfollows:\n“However, multiple lines of evidence indicate that the NGT domains lacks catalytic\nactivity (Fig. 3D, Figure 4A, Supplementary Fig. 5H). Consistently, our cryo-EM\nanalysis of the ternary complex further reveals that the acceptor peptide substrate binds\nspecifically to the CGT domain rather than the NGT domain (Fig. 4B). Additionally,\nalthough a negatively charged residue (D265) is located near the anomeric carbon (Fig.\n3B)—a position often associated with catalytic base function in glycosyltransferases—\nalanine substitution at this site only mildly impaired enzymatic activity (Fig. 3D),\nruling out its role in catalysis.”\n[3] Could authors clarify if MS experiments were run once per sample or were there\nrepeats.\nResponse: The MS experiments were run once per sample. We have clarified this in\nthe “Methods” section (Line 625). The objective of MS experiments was qualitative\nanalysis and we initially cross-validated MS findings with independent biochemical\nassays—for example, the consistent donor substrate specificity identified both in the\n the\nMS data (Fig. 1D-1E) and in the biochemical assays (Fig. 1C).\n[4] Lines 71-74: This part could better describe the motif e.g. presence of Hyp.\nResponse: We appreciate the reviewer’s suggestion. We have rephrased this part (Lines\n78-79 in the revised text) to better describe the motif, including the presence of\nhydroxylysine Hyl and hydroxyproline Hyp. It now reads as follows:\n“GLT25D1 is responsible for transferring galactose from UDP-galactose to\nhydroxylysine (Hyl) at specific Yaa-positions within the characteristic Gly-Xaa-Yaa\nmotifs of the collagen helical domain, where Yaa is often occupied by Hyl or Hyp\n(hydroxyproline).”\n[5] Line 96: It is unclear from the text which of the two elution peaks were used to\ndetermine that GLT25D1 was active.`\nResponse: The activity assay was conducted with the protein sample obtained after\naffinity chromatography and desalting, without size-exclusion chromatography. We\nhave clarified this in the “Methods” section (Lines 563-567).\nTo directly address the reviewer's query, we measured the enzymatic activities of\nboth elution peaks, and found that both peaks exhibit comparable levels of enzymatic\nactivity (Response Figure 3).\nResponse Figure 3. Enzymatic activity of GLT25D1 from two elution peaks.\nEnzymatic activity of GLT25D1 samples from two SEC size-exclusion\nchromatography peaks (10.3ml-peak and 11.8ml-peak, respectively).\n[6] Line 100: ‘In presence of Mn2+’ needs adding.\nResponse: We appreciate the reviewer’s suggestion and have revised accordingly\n(Lines 104-105 in the revised text).\n[7] Line122-126: Unclear if the protein used was a mix of the dimer/tetramer SE peak,\nor an isolated peak.\nResponse: Thank you for raising this important point. This comment is related to your\npoint #25. We have now supplemented the relevant details to address both issues.\nFor cryo-EM analysis of the apo-state (Supplementary Fig. 1A–C), we used a\npooled sample containing both major oligomeric forms, corresponding to the 10.3 mL\nand 11.8 mL SEC peaks shown in Figure 1B.\nFor cryo-EM analysis of the liganded ternary complex, since our data processing\nstrategy relied heavily on large numbers of dimer particles (Supplementary Fig. 6A–C),\nwe specifically used the later 11.8 mL SEC peak.\nWe have revised the indicated text (Lines 130–132; Line 245) as well as the\n“Methods” section (Line 630-631; Lines 634-635) to clearly state this information,\nensuring it is unambiguous for readers.\n[8] Line 172-174: How was significance determined? Also, by eye, it looks like activity\nof gelatin doesn’t change but does on Hyl containing peptide?\nResponse: We apologize for this confusion. The intended description was \"not\ndramatically\" rather than \"not significantly\". We have corrected this in the revised\nmanuscript (Line 181 in the revised text).\n[9] Line 180-190: Please include surface area for dimer-dimer interactions that\ncomprise the hexamer.\nResponse: Thank you for this valuable suggestion. We have included this surface area\n(1924 Å2) in the revised text (Line 190).\n[10] Line 242 space needed between ‘Fig.’ and ‘1D’.\nResponse: We have corrected this in the revised manuscript.\n[11] Line 251: Please specify in main text how H560 and R351 stabilise loop.\nResponse: We thank for this suggestion. In our initial submission, we had the\ndescription about H560 and R351 in the figure legend of Supplementary Fig. 7C. In the\nrevised manuscript, we have added this description to the main text (Line 255-257) as\nfollows:\n“H560 and R351 function to stabilize this ordered loop through backbone stacking at\nits beginning and a salt bridge with D570 at its middle, respectively (Supplementary\nFig. 7C)”\n[12] Line 324 “the SVD-associated point mutations L151R and A154P, located in helix\nα3, play critical roles in stabilizing the domain core fold”\nHere it is inferred the point mutations induce stability, which they don’t, I would\nrephrase and be more specific about the effects of point mutants, e.g. L151R is\nintroducing a larger, charged residue into a stabilising hydrophobic core.\nResponse: We thank the reviewer for this insightful suggestion. We have revised\naccordingly (Lines 330-335 under section “Molecular insights into pathogenic\nmutations of GLT25D1”). It now reads as follows:\n“The SVD-associated point mutations L151R and A154P, located in helix α3, are\nexpected to disrupt the domain's core fold stability (Fig. 6A). Specifically, the L151R\nmutation introduces a large, charged residue into a hydrophobic core, while A154P is\nlikely to impair helical structure and packing.”\n[13] Line 336 “Indeed, neither G377R nor R471W significantly”, while this is quite\npossibly the case, as band intensity has not been calculated and statistical tests not\nperformed, whether the result is ‘significant’ cannot be determined, the sentence needs\nto be less absolute.\nResponse: We appreciate this valuable suggestion and have revised accordingly. We\nhave removed the “significantly” and the new phrasing is: “neither G377R nor R471W\nappear to markedly affect …” (Line 345 in the revised text).\n[14] Line 375, β-pyrophosphate should be β-phosphate\nResponse: We have corrected \"β-pyrophosphate\" to \"β-phosphate\" (Line 385 in the\nrevised manuscript).\n[15] Line 456-467: Please provide more details regarding how the SDM was performed\nincluding primer sequences.\nResponse: We thank the reviewer for this suggestion. We have now provided a more\ndetailed description of the site-directed mutagenesis (SDM) procedure in the “Materials\nand Methods” section (Lines 529-536). Additionally, the corresponding primer\nsequences have been included as Supplementary Table 3.\n[16] Line 464: How were cells transferred to BMMY media.\nResponse: We thanks for this comment. In the revised manuscript, we have added\ndetailed description of the transferring to BMMY media as follows (Lines 540-542 in\n“Methods” section):\n“cells were harvested by centrifugation at 3,000g for 5 min. The BMGY medium was\nthen removed, and the cell pellet was resuspended in BMMY medium.”\n[17] Line 466: ‘following the manufacturer’s protocol’ it is unclear from the text which\nprotocol this is, as the sentence doesn’t refer to a very specific technique.\nResponse: We apologize for the confusion. The sentence referred to the standard\ninduction protocol from the Pichia Expression Kit USER GUIDE (Invitrogen,\nPublication Number MAN0000012). We have revised the manuscript to explicitly cite\nthis manual and include the specific induction conditions for clarity (Lines 542-545 in\n“Methods” section).\n[18] Line 469-470: ‘was applied to Ni-NTA affinity chromatography’ Ni-NTA is the\ntechnique, do you mean Ni-NTA resin? Was this gravity flow purification or FPLC-\nmediated?\nResponse: We thank the reviewer for this comment. In response, we have revised the\nphrasing to \"Ni-NTA resin\" as suggested and clarified that the purification was\nperformed using an ÄKTA pure chromatography system (Lines 548-550 in “Methods”\nsection).\n[19] Line 470: ‘After washing’, what was the wash buffer and how much was applied?\nResponse: We thank the reviewer for this comment. The washing procedure has been\ndescribed in detail in the revised manuscript (Lines 550-552 in “Methods” section),\nwhich now reads:\n\"The column was sequentially washed with 10 column volumes each of buffer A\ncontaining 20 mM, 50 mM, and 100 mM imidazole, respectively.\"\n[20] Line 472-472: What ratio of HRV3C to protein did you use? Where was the HRV3C\nsourced?\nResponse: We thank for this comment. The HRV3C protease (sourced from Genscript)\nwas used at a ratio of 1:20 (w/w, protease:protein), and this information has been\nincluded in the revised manuscript (Lines 553-556 in “Methods” section).\n[21] Line 478: How was protein concentration quantified? If by A280 please provide\next.co and Mw used.\nResponse: Thank you for this comment. Protein concentration was quantified by A280\nusing NanoDrop Spectrophotometers, with an extinction coefficient of 107635 M-1 cm-\n1 and a molecular weight of 68.3 kDa, respectively. These details have been added to\nthe revised manuscript in the “Methods” section (Lines 560-562).\n[22] Line 482: How was the hydroxylated peptide synthesised?\nResponse: We thank the reviewer for raising this question. We have now included the\ncommercial source of the hydroxylated peptide and a corresponding synthesis reference\nin the \"Methods\" section (Lines 571-575).\n[23] Line 484: I would prefer the concentration of protein to be reported in molarity to\nenable immediate comparison with the donor and acceptor substrate concentrations.\nResponse: We thank for this suggestion. As recommended, we have now included the\nprotein concentration expressed in both molarity and mass units (Lines 577-578 in the\n“Methods” section).\n[24] Line 526: How were grids glow discharged?\nResponse: We thanks for this comment. The detailed glow-discharge conditions have\nbeen added to the revised manuscript (Lines 628-629 in “Methods” section) as follows:\n\"The grids (Quantifoil R1.2/1.3 Au, 300 mesh) were glow-discharged using a PELCO\nEasiGlow unit at (15 mA for 60 s).\"\n[25] Line 525-532: It is unclear whether the dimer or tetramer peaks from the SE were\nused for the unliganded and ternary state. The latter cryo-EM data suggest tetramer\nwas used for the unliganded CTD structure and dimer for ternary structure, if this is\nnot the case, can authors comments on why they see dimer in the latter?\nResponse: Thank you for raising this important point. This comment is related to your\npoint #7. We have now supplemented the relevant details to address both issues.\nFor cryo-EM analysis of the apo-state (Supplementary Fig. 1A–C), we used a\npooled sample containing both major oligomeric forms, corresponding to the 10.3 mL\nand 11.8 mL SEC peaks shown in Figure 1B.\nFor cryo-EM analysis of the liganded ternary complex, since our data processing\nstrategy relied heavily on large numbers of dimer particles (Supplementary Fig. 6A–C),\nwe specifically used the later 11.8 mL SEC peak.\nWe have revised the indicated text (Lines 130–132; Line 245) as well as the\n“Methods” section (Line 630-631; Lines 634-635) to clearly state this information,\nensuring it is unambiguous for readers.\n[26] Line 563: Can the authors check the 275,468 number, a C3-symmetry expansion\nof 125,162 would suggest 375,486 particles.\nResponse: We apologize for the typographical error. The number should indeed be\n375,486 particles. We have corrected this.\n[27] Line 596: ‘Interactive’ not ‘manual’ rebuilding\nResponse: We have replaced ‘manual’ with ‘Interactive’.\n[28] Line 802: I think Figure 1A is a little confused as it doesn’t specifically show lysine\nresidues as the modified amino acids. I think a chemical diagram showing each step\nwould have been better but is not essential.\nResponse: Thank you for your valuable suggestion. We have revised Figure 1A\naccordingly. Chemical diagram showing the detailed modification on lysine has been\nadded (Fig. 1A).\nFig. 1A. Schematic representation of the collagen modification pathway.\nHydroxylysine residues formed by lysyl hydroxylases (LH/PLODs) at specific sites of\nnascent procollagen are galactosylated by GLT25D1/2. Some of this modification\nundergo further glucosylation by lysyl hydroxylases.\n[29] Line 838: Use red or pink to describe asterisk, not salmon as this is confusing.\nResponse: The term ‘salmon’ has been replaced with ‘pink’ to describe asterisk\naccordingly.\n[30] Line 857: Trimer not ‘timer’\nResponse: The spelling mistake has been corrected.\n[31] Line 870: The UDP in the diagram is all orange, making the phosphates difficult\nto ascertain and in contrast to the insert. I would change this to yellow to match insert.\nResponse: We thank for this suggestion. The UDP in Figure 3A and 3B has been\nchanged to yellow to match inset.\nFigure 3. Endogenous UDP-galactose bound to NGT domain of GLT25D1\n[32] Line 891: You are missing a few hydrogen bonds in this diagram suggested in main\ntext e.g. K470 to Ala in peptide.\nResponse: We thank for the suggestion. The hydrogen bonds (yellow dashed lines)\nbetween enzyme and peptide residues have been added into the revised Figure 5B.\nFigure 5B. Close-up view of the CGT active site in the ternary complex structure of\nGLT25D1 (in grey surface rendering) with collagen acceptor peptide (yellow stick) and\nUDP (cyan stick).\n[33] Line 913: For figure 6 the ‘C’ and ‘D’ are mapped incorrectly in legend, C should\nbe B and D should be C.\nResponse: The panel labels have been corrected as suggested.\n[34] Line 929-930 and 1001-1002: For Sup Figure 1D and E and 7D and E, please\nprovide the corrected, unmasked, masked, and randomised FSC curves.\nResponse: We thank the reviewer for this suggestion. In response, the updated\ncorrected, unmasked, masked, and randomised FSC curves have been provided.\n[35] Line 970: Trimer not ‘timer’\nResponse: The spelling mistake has been corrected.\n[36] Line 980-986: Can you provide uniport identifiers in parentheses after organism’s\nname in legend.\nResponse: We thank the reviewer for this suggestion. The corresponding uniport\nidentifiers have now been added in the specified place (legend of Supplementary Fig.\n4).\n[37] Line 1066: Most and least conserved not ‘conservative’.\nResponse: We thank the reviewer for this comment. \"Most/least conservative\" has been\nreplaced with \"most/least conserved\" in the specified place (legend of Supplementary\nFig. 9).\nReviewer #5 (Remarks to the Author):\nThis manuscript presents a rigorous structural and biochemical dissection of GLT25D1,\na collagen galactosyltransferase essential for post-translational modification of\nhydroxylysine residues. The authors employ high-resolution cryo-EM, structure-guided\nmutagenesis, and enzymatic assays to map the catalytic site, elucidate substrate\nspecificity, and rationalize the effects of disease associated mutations. The work is\ntechnically sound and clearly presented, and it provides a valuable resource for\nunderstanding the molecular mechanism of collagen glycosylation.\nHowever, in considering the broader impact expected for publication in Nature\nCommunications, I am uncertain whether the conceptual advance reaches the\nnecessary threshold. While the structural insights into GLT25D1 are new and welcome,\nthey pertain to a single, well-known enzyme, and the study largely confirms or refines\nprior expectations (e.g., involvement of the CGT domain in catalysis, recognition of the\n“X-Hyl-Gly” motif). The physiological or translational implications are discussed but\nnot experimentally explored, and the potential connections to disease, though\ninteresting, remain somewhat speculative.\nResponse: We thank the reviewer for the positive assessment of our study’s technical\nquality and value to the field of collagen glycosylation. We have carefully considered\nthe comment and have revised the Discussion to more clearly present the unique\ncontribution and the broader implications of our findings.\nAlthough GLT25D1 is a well-known important enzyme in collagen biology and is\nlinked to severe human diseases (such as SVDs, VCI, connective tissue disorders),\nthe molecular basis underlying its crucial collagen modifying activity has remained\nelusive. Our work addresses this key gap by resolving the first ternary complex of\nGLT25D1 with both UDP/UDP-galactose and a designed hydroxylysine-containing\ncollagen-mimetic substrate. Such a ternary complex is essential for understanding a\nprotein-targeting glycosyltransferase like GLT25D1. This advance, complemented by\ncomprehensive functional analyses, enables us not only to unambiguously delineate the\ncatalytic center within the CGT domain and elucidate the molecular determinants\ngoverning collagen recognition, but also to establish an inverting S 2 catalytic\nN\nmechanism with Asp522 acting as the catalytic base.\nBeyond these insights, our work also reveals several important and unexpected\nfindings. We identified an endogenous UDP-galactose molecule bound to the non-\ncatalytic NGT domain that contributes to structural stability, suggesting a previously\nunrecognized mode of enzyme regulation. Furthermore, we uncovered evidence of a\nhigher-order oligomeric assembly (dimer–hexamer), an architectural feature not\npreviously described in this enzyme family. Finally, by systematically characterizing\ndisease-associated mutations linked to SVDs, VCI, and related connective tissue\ndisorders, we provide mechanistic explanations for how disease-associated mutations\nimpair GLT25D1 function through distinct molecular mechanisms. These insights not\nonly advance the molecular understanding of disease etiology but also establish a\nfoundation for developing biochemical diagnostics to detect GLT25D1 dysregulation.\nIn response to Reviewer #1’s suggestion, we have further expanded the Discussion to\ninclude potential therapeutic strategies targeting GLT25D1 dysfunction.\nCollectively, these results establish a comprehensive mechanistic framework for\ncollagen galactosylation that spans donor and acceptor substrate recognition, precise\ncatalytic mechanism, regulation, oligomerization architecture, and pathogenic\nmechanisms. We hope that this work will stimulate future research to explore the\nfundamental and translational implications of these findings.\nMinor comments:\nLine 73: the authors refer to a Gly-X-Y sequence. Y is the symbol for tyrosine, but it\nseems it is not used to denote tyrosine here.\nResponse: We thank for this comment. To avoid confusion of symbol Y, ‘Gly-X-Y’\nhave been replaced with three-letter symbol ‘Gly-Xaa-Yaa’ (Line 78 in the revised text).\nThere are several instances where \"timer\" has been written in place of \"trimer\" by\nmistake.\nResponse: We thank for this suggestion. The spelling mistakes have been corrected.\nPoint-to-point responses to the reviewers’ comments\nWe greatly thank the constructive suggestions from reviewers. Our responses below are\nin blue.\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nThe authors have done a great job and have thoroughly addressed my previous\ncomments. I have only two minor points for further clarification:\nResponse: We thank the reviewer for the support and for the valuable help to improve\nour manuscript.\na.-) Could the authors explain why the CGT domain exhibits a better Km towards\ngelatin than the wild-type enzyme? It seems the NGT domain may negatively affect\nbinding to gelatin.\nResponse: We thank the reviewer for this insightful comment. We agree with the\nreviewer that CGT domain exhibits a lower Km than the WT enzyme, suggesting that\nNGT domain may negatively affect the binding of gelatin to CGT domain. This\ninterpretation is consistent with our kinetic data and molecular dynamics simulations,\nwhich indicate allosteric communication between the NGT and CGT domains\n(Supplementary Fig. 11). To clarify this point, we have added the following statement\nin the revised ‘Discussion’ section (fourth paragraph):\n“Consistent with this notion, kinetic analysis also indicates that the NGT domain\ninfluences the CGT domain, as evidenced by a lower Km of the CGT domain toward\ngelatin compared to that of the wild-type enzyme (Supplementary Fig. 11A).”\nb.-) How many replicates were performed for the molecular dynamics simulations?\nI have not found this in Methods.\nResponse: We thank the reviewer for pointing this out. The molecular dynamics\nsimulations were conducted with four replicates. This missing information has now\nbeen added to the “Molecular dynamics simulations” subsection (second paragraph)\nwithin the Methods section of the revised manuscript.\nReviewer #2(Remarks to the Author):\nDear authors, fellow reviewers, and editorial team,\nThe authors have addressed the concerns I had in my original review. I'm particularly\nglad to see that Tyr flipping into the density in a visually pleasing fashion.\nI lack the subject specific knowledge to comment on the other reviewers comments.\nYours sincerely,\nJamie Blaza\nUniversity of York\nResponse: We thank the reviewer for the support and for the valuable help to improve\nour manuscript.\nReviewer #3(Remarks to the Author):\nThe authors have answered all my concerns. I am now recommend the publication of\nthis work.\nResponse: We thank the reviewer for the support and for the valuable help to improve\nour manuscript.\nReviewer #4 (Remarks to the Author):\nThe authors have made considerable improvements to the manuscript since the initial\nreview improving its overall quality. The results support the conclusions, and the results\nare of significance to the field. The methodology is sound and meets the expectations of\nthe field and is sufficiently detailed to be able to be reproduced. My points raised within\nthe initial review have been addressed and the work is now of high quality.\nResponse: We thank the reviewer for the support and for the valuable help to improve\nour manuscript.\nAlthough, I have three very minor edit suggestions, linked to the new edits made:\n[1] Line 181 in revised manuscript needs to say \"against gelatin\" as the peptide with\nHyl is significantly less.\nResponse: We thank the reviewer for this valuable suggestion and we have revised\naccordingly by adding ‘against gelatin’ as suggested.\n[2] Line 1050 p value number is same for***and****, they should be different.\nResponse: We apologize for the typo. We have corrected this (in the figure legend of\nFigure 2I) and it now reads: “***p < 0.001; ****p < 0.0001”.\n[3] Line 1094 the hydrogen bonds differ between \"B” and \"D\" even though they are\nsimply rotated images, this likely results from incorrect H-bonds in \"B”. Tied to this\nLine 298-299\"its interaction with acceptor peptide only relies on the B-carbon of this\nresidue\" while in fact H-bonding suggests involvement of T562 hydroxyl. Did the\nauthors mean \"sidechain\"?\nThese edits are minor and do not detract from my enthusiasm for the work.\nResponse: We thank the reviewer for the support and for this insightful comment. We\nwould like to clarify that panels “B” and “D” illustrate different aspects of the\ninteraction. While panel “B” highlights specific hydrogen bonds, panel “D” displays\ndistances between selected groups to depict the narrowest constriction within the\nbinding cleft. We have revised the sentence noted by the reviewer to avoid confusion\n(located in the first paragraph under section ‘Acceptor peptide binding mode and\ncatalytic mechanism’). The revised sentence now reads as follows:\n“Interestingly, alanine substitution of T562 preserved activity (Fig. 5E), suggesting that\nits interaction with acceptor peptide may more rely on the β-carbon of this residue (Fig.\n5D).”\nReviewer #5(Remarks to the Author):\nAll of my comments have been addressed in the revision.\nResponse: We thank the reviewer for the support and for the valuable help to improve\nour manuscript."}]

Decision letter