# Uncommon N-Glycan Structures in Anhydrobiotic Tardigrades

H. Yagi, T. Saito, S. Y. Guu, Nao Yamakawa, S. Shimamura, S. Kondo, M.

Yagi-Utsumi, K. Takai, J. I. Furukawa, Yann Guerardel, et al.

► To cite this version:

H. Yagi, T. Saito, S. Y. Guu, Nao Yamakawa, S. Shimamura, et al., Uncommon N-Glycan Structures in Anhydrobiotic Tardigrades. Molecular and Cellular Proteomics, 2025, Molecular and Cellular Proteomics, 24 (6), pp.100979. 10.1016/j.mcpro.2025.100979. hal-05152321

HAL Id: hal-05152321

https://lilloa.hal.science/hal-05152321v1

Submitted on 9 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Uncommon *N*-Glycan Structures in Anhydrobiotic Tardigrades

## Authors

Hirokazu Yagi, Taiki Saito, Shih-Yun Guu, Nao Yamakawa, Shigeru Shimamura, Sachiko Kondo, Maho Yagi-Utsumi, Ken Takai, Jun-ichi Furukawa, Yann Guérardel, Kay-Hooi Khoo, Kazuharu Arakawa, and Koichi Kato

## Correspondence

kkatonmr@ims.ac.jp

## In Brief

This study identifies paucimannose-type glycans as dominant *N*-glycosylation and uncommon *N*-glycans with Fucα1,3-GlcNAc motifs in anhydrobiotic tardigrades. These Fucα1,3-GlcNAc structures are associated with proteins involved in oxidative stress protection and extracellular matrix remodeling. Genomic analyses suggest specific fucosyltransferases responsible for this glycosylation. These findings offer insights into the molecular mechanisms underlying tardigrade resilience and highlight the potential role of glycans in surviving extreme stress.

## Graphical Abstract

<figure><img src="image_2.png" /></figure>

Tardigrade

## Highlights

* Paucimannose-type glycans dominate *N*-glycosylation in tardigrades.

* Uncommon antennal Fucα1,3-GlcNAc structure is prevalent in *Hypsibius exemplaris*.

* Fucα1,3-GlcNAc motif is found in certain glycoproteins induced during anhydrobiosis.
# Uncommon *N*-Glycan Structures in Anhydrobiotic Tardigrades

Hirokazu Yagi¹,², Taiki Saito¹,² ID, Shih-Yun Guu³ ID, Nao Yamakawa⁴,⁵,⁶ ID,
Shigeru Shimamura⁷ ID, Sachiko Kondo¹,², Maho Yagi-Utsumi¹,² ID, Ken Takai⁷,
Jun-ichi Furukawa⁶ ID, Yann Guérardel⁵,⁸ ID, Kay-Hooi Khoo³, Kazuharu Arakawa²,⁹,¹⁰ ID,
and Koichi Kato¹,²,¹¹,*

We characterized the *N*-glycosylation profiles of anhydrobiotic tardigrades, *Ramazzottius varieornatus* and *Hypsibius exemplaris*, identifying high-mannose, paucimannose, and complex-type oligosaccharides, while hybrid-type glycans were undetectable. Notably, paucimannose-type oligosaccharides accounted for 39% of the *N*-glycans in *R. varieornatus* and 17% in *H. exemplaris*, with a substantial proportion of them exhibiting fucosylation of the innermost GlcNAc via an α1,6-linkage. This core fucosylation pattern, common to all animals, was observed alongside a distinctive glycosylation signature prominently observed in tardigrades: complex-type glycans lacking galactosylation but containing α1,3-fucosylated GlcNAc at non-reducing termini. This structure was more prevalent in *H. exemplaris*, with 22 out of 87 identified glycoproteins expressing the Fucα1,3-GlcNAc motif, including eight induced during anhydrobiosis. Key glycoproteins such as Cu/Zn-superoxide dismutase and papilin, implicated in oxidative stress protection and extracellular matrix remodeling, were among those modified. Comparative analyses reveal that non-reducing terminal α1,3-fucosylation in tardigrades is distinct from the mammalian Lewis X antigen and similar structures found in invertebrates, suggesting a unique substrate specificity of fucosyltransferases in these species. Genomic analysis identified homologs of FUT9 and FucTC, indicating potential candidates responsible for this glycosylation pattern. Our findings provide new insights into the molecular mechanisms of glycosylation in tardigrades and their relevance to their extreme stress tolerance.

Tardigrades are members of the superphylum Ecdysozoa, which includes molting invertebrates (1). Over 1400 species of

tardigrades have been discovered in various environments such as the sea, land, and lakesides, thriving under wet conditions (2). Many terrestrial tardigrades can adapt to dry conditions through anhydrobiosis, a state where metabolic activity ceases due to extreme water deprivation. In this anhydrobiotic state, tardigrades exhibit remarkable tolerance to various extreme environmental conditions, including extreme temperatures, radiation, vacuum, and toxic chemicals (3–7). This process is reversed, and they immediately return to their normal metabolic state upon rehydration.

In recent years, significant progress has been made in understanding the molecular mechanisms underlying tardigrade stress tolerance. The establishment of standard culture systems for species such as *Ramazzottius varieornatus* and *Hypsibius exemplaris* has facilitated genetic and biochemical analyses (8, 9). Several proteins have been identified that are believed to play key roles in anhydrobiosis and stress tolerance, including cytoplasmic abundant heat soluble (CAHS) proteins, secretory abundant heat soluble (SAHS) proteins, mitochondrial late embryogenesis abundant (LEAM) proteins, and mitochondrial abundant heat soluble (MAHS) proteins (7, 10–13). However, the precise mechanisms governing anhydrobiosis in tardigrades remain to be fully elucidated.

*N*-Glycosylation is a major post-translational modification that regulates cell-cell communication among various cell types. *N*-glycans in eukaryotes share a common core structure consisting of three mannose (Man) and two *N*-acetylglucosamine (GlcNAc) residues, with species-specific structures at the non-reducing ends. *N*-glycan structures vary in size, components, and branching patterns, reflecting the

From the ¹Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan; ²Department of Creative Research, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki, Japan; ³Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; ⁴Université de Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, US 41-UAR 2014-PLBS, Lille, France; ⁵Université de Lille, CNRS, UMR 8576 - UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France; ⁶Institute for Glyco-Core Research, Nagoya University, Nagoya, Japan; ⁷Institute for Extra-cutting-edge Science and Technology Avant-garde Research (X-star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan; ⁸Institute for Glyco-Core Research, Gifu University, Gifu, Japan; ⁹Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan; ¹⁰Graduate School of Media and Governance and Faculty of Environment and Information Studies, Keio University, Fujisawa, Kanagawa, Japan; ¹¹Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Japan

<footnote>*For correspondence: Koichi Kato, kkatonmr@ims.ac.jp.</footnote>
organism's physiological response to its environment. In tardigrades, these *N*-glycans are likely to contribute to their extraordinary survivability in extreme environments by mediating various cellular processes.

In this study, we report the detailed *N*-glycan profiles of two closely related tardigrade species, *H. exemplaris* and *R. varieornatus* (both members of the superfamily Hypsibioidea). Our analysis reveals the presence of distinctive glycan structures in these tardigrades. Additionally, we identified specific glycoproteins bearing these carbohydrate moieties, some of which are known to be induced under desiccation conditions.

## EXPERIMENTAL PROCEDURES

### Tardigrade Culture

The tardigrades, *H. exemplaris* and *R. varieornatus*, were cultured as previously described (8, 14). In summary, they were nourished with *Chlorella vulgaris* (Chlorella Industry) on 2% Bacto Agar (Difco) plates and maintained at 18 °C for *H. exemplaris* and 22 °C for *R. varieornatus* under dark conditions. Anhydrobiotic adult specimens were placed within a controlled chamber with relative humidity gradients. The criterion for successful anhydrobiosis was set at >90% recovery rate after rehydration, as observed across samples prepared within the same chamber.

### Preparation of Fluorescence-Tagged *N*-glycans

All experimental procedures for profiling, including chromatographic conditions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MS/MS techniques, have been previously described (15–17).

*N*-glycans were released from glycoproteins obtained from 6000 tardigrades in an anhydrobiotic state through liquid-phase hydrozinolysis, followed by pyridylamination. Initially, the dried material was sequentially extracted three times using chloroform/methanol mixtures in ratios of 2:1 (v/v) and 1:2 (v/v). The pellet fraction, which contained the glycoproteins, was then resuspended in 20 mM Tris-HCl buffer (pH 8.0) and centrifuged at low speed to remove insoluble debris. The resulting supernatant was precipitated by adding 70% ethanol at 4 °C and incubating overnight. The precipitate was collected by centrifugation at 1200 g as a protein pellet.

This protein pellet was dried using a rotary vacuum evaporator and subsequently incubated with 200 µl of hydrazine anhydride in a screw-capped glass tube at 95 °C for 10 h to release the glycans. The reaction was quenched by mixing with 3 ml of 50 mM ammonium acetate buffer (pH 7.0), and then the mixture was loaded onto a graphite carbon column (GL-Pak Carbograph 150 mg/3 ml, GL Sciences) that had been pre-equilibrated with the same buffer. After washing the column with 5 ml of ammonium acetate buffer and 5 ml of triethylamine acetate buffer (40% acetonitrile, pH 7.0), the glycans were eluted using 5 ml of triethylamine acetate buffer containing 2% acetic anhydride. The eluate was dried using a rotary vacuum evaporator to yield *N*-acetylated glycans.

For pyridylamination, a labeling solution was prepared by dissolving 276 mg of 2-aminopyridine in 100 µl of acetic acid. The dried glycan sample was dissolved in 30 µl of the labeling solution and incubated at 90 °C for 60 min in a tightly sealed screw-capped glass tube. Subsequently, a reducing solution, consisting of 200 mg of dimethylamine-borane dissolved in a mixture of 80 µl acetic acid and

50 µl ultrapure water, was added to the reaction tube. The mixture was incubated at 80 °C for 35 min to complete the reaction.

To purify the pyridylaminated (PA) glycans, the reaction mixture was applied to a cellulose column equilibrated with buffer I (66% 1-butanol, 16% ethanol, 16% 0.6 M acetic acid). The column was washed twice with the same buffer before glycans were eluted with 2 ml of buffer II (33% ethanol, 66% 75 mM ammonium bicarbonate). The eluate was then dried using a SpeedVac vacuum concentrator, yielding purified PA-labeled glycans suitable for further analysis. The resulting PA-glycans were then dried, redissolved in double-distilled water, and subjected to HPLC analysis.

### Fractionation and Structural Analysis of PA-Glycans by HPLC

The PA-glycan sample mixture was fractionated using reverse-phase HPLC with a Shim-pack HRC octadecyl silica (ODS) column (6.0 mm inner diameter x 150 mm; Shimadzu, Kyoto, Japan). PA-labeled glycans were detected by fluorescence at excitation and emission wavelengths of 320 nm and 400 nm, respectively. Elution was performed at a flow rate of 1.0 ml/min at 55 °C using two solvents: A and B. Solvent A was 10 mM sodium phosphate buffer (pH 3.8), and solvent B was 10 mM sodium phosphate buffer (pH 3.8) containing 0.5% 1-butanol. The column was equilibrated with 80% solvent A and 20% solvent B. The gradient elution was set from 0 to 60 min with a linear gradient of 20 to 50% solvent B. PA-labeled glucose oligomers (Takara Bio Inc., Shiga, Japan), composed of glucose units linked via α-1,6-glycosidic bonds with defined degrees of polymerization (DP4–DP21), were analyzed in advance using ODS columns. The elution times were converted to glucose units (GU), which reflect the degree of polymerization. This conversion allows for normalization of retention times, compensating for lot-to-lot variation and column degradation, thereby ensuring analytical consistency.

Each PA-glycan fraction was separated based on differences in ODS elution time, attributed to the intrinsic chemical structure of the glycans. Each fractionated PA-glycan sample was mixed with a matrix solution (10 mg/ml dihydrobenzoic acid) and analyzed using MALDI-TOF-MS on an Autoflex Speed instrument (Bruker Daltonics) in positive-ion reflector mode. For MS acquisition, the PA glycans in water were mixed with dihydrobenzoic acid (10 mg/ml in 50% acetonitrile) at a 1:1 ratio, and the resulting mixture was spotted onto the target plate. The instrument settings included a laser intensity of 50 to 78%, resulting in a total of 5000 shots for linear positive mode MS. The MS survey and data acquisition were performed manually. By comparing the mass values of PA-glycans and their elution positions on the ODS column with data in the GALAXY database (18, 19), the structure and/or components of each *N*-glycan fraction were anticipated. This method enables the identification of glycan isomers, making the resulting data essential for interpreting the glycoproteomics data obtained from the nanoLC-MS/MS analysis of glycopeptides described below. PA-glycans that did not match any of the previously registered *N*-glycans in the GALAXY database were trimmed using α-fucosidase from bovine kidney (Sigma), α-mannosidase (Seikagaku Co.), and/or β-*N*-acetylglucosaminidase S, which was cloned from *Streptococcus pneumoniae* and expressed in *E. coli*. (New England Biolabs), to align their structures with known forms. To identify the PA-glycans in peaks 8 and 11, MS/MS analysis was conducted in positive ion mode using a Bruker UltrafléXtreme MALDI-TOF/TOF mass spectrometer. The analysis employed LIFT mode under reflector positive ion conditions. The acquired mass spectra were processed using flexAnalysis 3.4 software (Bruker Daltonics).

### MALDI-TOF-MS and MS/MS Analysis of Permethylation of *N*-glycans

PA-glycans with mass values indicating the presence of fucose and *N*-acetylglucosamine residues at the non-reducing end, but lacking
galactose residues, were further permethylated using the NaOH/dimethyl sulfoxide slurry method as previously described (15, 16). The permethylated sample aliquots were mixed 1:1 with matrix solution of 10 mg/ml 2,5-dihydroxybenzoic acid in 50% acetonitrile for positive mode analysis, and then spotted onto the MALDI plate for data acquisition using an AB SCIEX MALDI TOF/TOF 5800 system. The instrument settings included a laser intensity of 4500 with 50 subspectra, each consisting of 100 shots, resulting in a total of 5000 shots for reflector positive mode MS1. For MS/MS analysis in reflector positive mode at 2 kV CID energy, the laser intensity was set to 5000 under the same subspectral conditions. To enhance the signal-to-noise ratio, multiple MS/MS spectra of the same precursor were combined to generate the final spectrum. The mass resolution for the MS mode was approximately 16,000 for peaks around m/z 1500 and 5000 for MS2 fragment ions around m/z 1000. The precursor mass window was configured at 200 resolution (FWHM), with the metastable suppressor activated.

The MS survey and data acquisition were conducted manually. All permethylated glycans and glycan fragments were identified as [M+Na]⁺ sodium adducts. Specific MS ions were selected as parent ions for MS/MS fragmentation pattern acquisition after examining the summed MALDI-TOF-MS spectra (15, 16).

## Gas Chromatography-Electron Impact MS (GC-EI-MS) Methylation Analyses

Approximately 200 pmol of PA-labeled glycan mixtures from *H. exemplaris* and *R. varieornatus* were permethylated using the NaOH/dimethyl sulfoxide slurry method, as described in the previous study (20). The permethylated oligosaccharides were hydrolyzed with 4 M trifluoroacetic acid at 100 °C for 4 h, followed by reduction with 10 mg/ml sodium borodeuteride (NaBD₄) for 16 h at room temperature. Subsequently, the resulting products were acetylated to yield the partially methylated alditol acetates (PMAA).

Gas chromatography (GC) was performed on a Trace GC Ultra system (Thermo Scientific) equipped with a SOLGEL-1MS capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness) and detected by a TSQ Quantum GC detector (Thermo Scientific). The carrier gas was helium (99.9995%) at a constant pressure of 100 kPa and an oven temperature of 120 °C. The temperature gradient was set from 120 °C to 230 °C over 55 min at a rate of 2 °C/min, and then increased to 270 °C in 4 min at a rate of 10 °C/min. An electron ionization source mode at 70 eV was used for the mass spectrometer operation. The mass spectral data and retention times of the PMAA derivatives of 4-GlcNAc, 3-GlcNAc, and non-reducing terminal GlcNAc were compared with those of authentic standard compounds for identification.

## NanoLC-MS Measurements of Tardigrade Glycopeptides

For the identification of glycoproteins carrying a specific glyco-tope, Fucα1,3-GlcNAc, we conducted nanoLC-MS/MS analysis on protein fractions derived from *H. exemplaris*. Proteins were extracted from approximately 10,000 tardigrades, alkylated, and digested with trypsin. The extraction involved sequentially treating the dried material with its lipid was sequentially extracted from the cells with 80% ethanol, 100% ethanol, chloroform/methanol (2:1, v/v), chloroform/methanol/H₂O (1:2:0.8, v/v/v), and 80% acetone, as previously described (21). The proteins were then dissolved in 500 µl of Milli-Q water, denatured with 0.1% Rapigest (Waters) in 50 mM Tris-HCl, reduced with DTT (Fujifilm Wako), alkylated with iodoacetamide (Fujifilm Wako), and digested with Lys-C (Fujifilm Wako) at a ratio of 1:200 (weight of protein) and sequencing-grade modified trypsin (Promega) at a ratio of 1:100 (weight of protein) at 37 °C overnight in 0.1% Rapigest. After digestion, Rapigest was removed by acidification with 0.1% trifluoroacetic acid (TFA) (Fujifilm Wako).

The resulting peptides were then loaded onto an amide column (TSK-gel Amide-80, 2 × 50 mm; TOSOH) to enrich glycopeptides,

<figure><img src="image_3.png" /><figcaption>FIG. 1. HPLC N-glycan profiles on the ODS column derived from *R. varieornatus* and *H. exemplaris*. The peaks were numbered in order of elution time. PA-oligosaccharides were assigned using Glycomod (https://web.expasy.org/glycomod/) with a mass tolerance of ±0.5 Da. Peaks unassignable to N-glycans are indicated by asterisks.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>FIG. 2. MALDI MS/MS sequencing of the permethylated PA-glycans. The permethylated PA-glycans found in fractions (A) 16, (B) 17, (C) 18, and (D) 19 were subjected to MS/MS fragmentation analysis. Each of the sodiated molecular ions afforded a series of sodiated reducing end ¹,⁵X and Y ions, which collectively established their overall sequences. The non-reducing terminal units, either a HexNAc or a FucHexNAc, were identified by the oxonium ions at m/z 260 or 434 (annotated in red), respectively. The latter was additionally represented by sodiated B and E ions at m/z 456 and 385 (also annotated in red). The fragment ions unique to high energy CID cleavages on a MALDI TOF/TOF, including the E, G, and</figcaption></figure>
<table><caption>TABLE 1
GC-MS linkage analysis of partially methylated alditol acetates prepared from the PA-glycans derived from R. varieornatus and H. exemplaris</caption><thead><tr><th rowspan="2">O-methylated alditol acetates<br>(glycosyl residue)<sup>a</sup></th><th colspan="2">Elution time (min)</th><th rowspan="2">Characteristic<br>fragment ions (m/z)</th><th colspan="2">Relative abundance<sup>b</sup></th></tr><tr><th>R. varieornatus</th><th>H. exemplaris</th><th>R. varieornatus</th><th>H. exemplaris</th></tr></thead><tbody><tr><td>2,3,6-O-trimethyl-GlcNAc-ol (4-GlcNAc)</td><td>35.54</td><td>38.50</td><td>75, 99, 117, 143, 159, 233</td><td>1.00</td><td>1.00</td></tr><tr><td>2,4,6-O-trimethyl-GlcNAc-ol (3-GlcNAc)</td><td>40.49</td><td>40.44</td><td>75, 100,117, 129,<br>159, 161, 275</td><td>0.07</td><td>0.17</td></tr><tr><td>2,3,4,6-O-tetramethyl-GlcNAc-ol<br>(Terminal-GlcNAc)</td><td>35.20</td><td>35.15</td><td>75, 117, 129, 145, 159,<br>161, 203, 205</td><td>0.88</td><td>0.88</td></tr><tr><td>2,3,4-O-trimethyl-Fuc-ol (Terminal-Fuc)</td><td>16.94</td><td>16.96</td><td>59, 102, 115, 118, 131, 175</td><td>0.06</td><td>0.10</td></tr></tbody></table>

<sup>a</sup>The glycosyl residues were identified based on the relative retention time and characteristic EI-MS fragment ions afforded by the respective partially methylated alditol acetates, with reference to authentic standards.

<sup>b</sup>The respective TIC peak areas were integrated for an approximate indication of their abundance relative to 4-GlcNAc, which was arbitrarily set as 1.00, without correcting for individual response factors.

following the method outlined in a previous study (22). Glycopeptides from tryptic digests were captured using an Amide-80 column that had been equilibrated with 75% acetonitrile (Merck) and 0.1% TFA and were subsequently eluted isocratically with 50% acetonitrile and 0.1% TFA after washing.

The glycoprotein fraction was then analyzed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) coupled with an UltiMate 3000 RSLCnano Nanoflow liquid chromatography system (Thermo Fisher Scientific). Glycopeptide separations were performed on a reverse-phase column (Zaplous alpha Pep-C18, 3 µm, 120 Å, 0.1 × 150 mm. AMR Inc) using a linear gradient of 5%-45% solvent B over 220 min at a constant flow rate of 500 nl/min. Solvent A was 0.1% formic acid, and solvent B was 100% acetonitrile. A nano-electrospray ion source (Dream spray, AMR Inc.) was used, with the column oven set to 35 °C.

The Orbitrap Fusion Tribrid mass spectrometer operated in positive ion mode with an electrospray voltage of 1.6 kV and an ion transfer tube temperature of 250 °C. Full scan MS spectra were acquired in the Orbitrap mass analyzer (m/z range: 350-1800, resolution: 120,000 FWHM) using internal calibration (EASY-IC). MS/MS spectra were obtained in the Orbitrap mass analyzer (mass range: normal, scan range mode: auto, resolution: 50,000 FWHM) using higher-energy collisional dissociation (HCD) with a normalized collision energy of 28% for efficient detection of signature ions. The HCD fragment ions corresponding to glycan oxonium fragments (HexNAc: m/z 204.0867, HexNAc-Hex: m/z 366.1396, HexNAc-DHex: m/z 350.1395, HexNAc fragment: m/z 138.0545) were used to trigger EThcD/CID fragmentation. When glycan signature ions were detected in the HCD spectrum within 15 ppm mass accuracy, additional precursor isolation and MS/MS spectra were acquired using two methods:

1. The Ion Trap mass analyzer (m/z range: auto, scan rate: rapid) with 35% CID energy.

2. The Orbitrap mass analyzer (mass range: normal, scan range mode: define first mass m/z 120, resolution: 50,000 FWHM) with 25% EThcD collision energy.

## Data Analysis for Identification of Glycoproteins

Glycopeptide analysis was performed using Byonic v5.7.11 (Protein Metrics Inc., Cupertino, CA, USA) as previously described (23). Briefly, the acquired spectra were searched against a database of coding sequences identified in the genome of *H. exemplaris* (Proteome ID UP000192578 in UniProt). Searches were performed with the following parameters: the precursor mass tolerance was 10 ppm; the fragment mass tolerance was 20 ppm; trypsin as the digestion enzyme with a maximum of two missed cleavages; carbamidomethylation of cysteine as a fixed modification; oxidation of methionine as a variable modification; targeted FDR (strict) for PSMs was 1%. N-glycan modifications were assessed using the N-glycan 38 insect library supplemented with the following glycans: HexNAc (4)Hex(3)Fuc(3), HexNAc(3)Hex(3)Fuc(2), HexNAc(2)Hex(3)Fuc(1), HexNAc(5)Hex(3) Fuc(2), and HexNAc(4)Hex(3)Fuc(2). Glycopeptide spectral matches were stringently filtered, requiring a Byonic score ≥200 and ILog Probl ≥ 2 to ensure high-confidence identifications. The output data from the Byonic search is presented in Supplemental Table S1, as it provides information on the glycopeptides and glycoproteins identified in the LC-MS/MS analysis.

## BLAST Analysis

Protein sequence similarity searches were conducted using the NCBI BLAST + tool (version 2.15.0), available at https://blast.ncbi.nlm.nih.gov/. The query protein sequences were aligned against the nr database (non-redundant protein sequences; downloaded January 2024). Searches were performed using BLASTp with the following parameters: an e-value threshold of 1e-5, a word size of 3, and default gap penalties. Taxonomic filters were applied to focus the search on *H. exemplaris*. The results were analyzed based on the Bit score, which normalizes the score of sequence alignments, to enable comparability across searches. Only alignments with a Bit score of ≥50 were considered, as this threshold effectively eliminated low-confidence matches.

D ions, have been described previously (36) and were named accordingly. The D ions (annotated in blue) allow defining the 6-arm substituents on the trimannosyl core although alternative isomeric arrangements could not be ruled out. The cartoon drawings of the assigned glycan structures follow the recommended symbol nomenclature for glycans (37). The structures of the PA-glycans from fractions 16, 17, 18, and 19 were assigned based on MALDI MS/MS sequencing analysis as well as HPLC mapping combined with treatments using β-N-acetylhexosaminidase and/or α-fucosidase.
<table><caption>TABLE 2
N-Glycans derived from R. varieornatus and H. exemplaris</caption><thead><tr><th rowspan="2">Peak</th><th rowspan="2">GU (ODS)<sup>a</sup></th><th rowspan="2">Observed mass value<sup>b</sup></th><th rowspan="2">Structure</th><th colspan="2">Relative quantity<sup>c</sup> (mol %)</th></tr><tr><th>R. varieornatus</th><th>H. exemplaris</th></tr></thead><tbody><tr><td>1</td><td>5.0</td><td>1821.6</td><td>Manα2Manα6, Manα3, Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>4.2</td><td>5.5</td></tr><tr><td>2</td><td>5.2</td><td>1983.7</td><td>Manα2Manα6, Manα3, Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>14.8</td><td>10.5</td></tr><tr><td>3</td><td>5.8</td><td>1659.7</td><td>Manα6, Manα3, Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>-</td><td>1.2</td></tr><tr><td>4</td><td>6.1</td><td>1497.5</td><td>Manα6, Manα3, Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>3.8</td><td>6.7</td></tr><tr><td>5</td><td>6.3</td><td>2145.6</td><td>Manα2Manα6, Manα3, Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>1.8</td><td>3.2</td></tr><tr><td>6</td><td>7.1</td><td>1335.4</td><td>Manα6, Manα3, Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>7.1</td><td>3.9</td></tr><tr><td>7</td><td>7.4</td><td>849.3</td><td>Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>1.5</td><td>-</td></tr><tr><td></td><td></td><td>1011.3</td><td>Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>3.7</td><td>2.8</td></tr><tr><td></td><td></td><td>1173.4</td><td>Manα3, Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>4.9</td><td>1.1</td></tr><tr><td></td><td></td><td>1214.4</td><td>Manα6, Manβ4GlcNAcβ4GlcNAc</td><td>1.2</td><td>2.0</td></tr><tr><td>8<sup>d</sup></td><td>8.4</td><td>979.4</td><td>Fucα6, Manβ4GlcNAcβ4GlcNAc</td><td>2.4</td><td>-</td></tr></tbody></table>
<table><caption>TABLE 2—Continued</caption><thead><tr><th rowspan="2">Peak</th><th rowspan="2">GU (ODS)<sup>a</sup></th><th rowspan="2">Observed mass value<sup>b</sup></th><th rowspan="2">Structure</th><th colspan="2">Relative quantity<sup>c</sup> (mol %)</th></tr><tr><th>R. varieornatus</th><th>H. exemplaris</th></tr></thead><tbody><tr><td>9</td><td>8.8</td><td>1141.4</td><td>Manα6、<br>Manβ4GlcNAcβ4GlcNAc<br>Fucα6、<br>Fucα3</td><td>0.7</td><td>-</td></tr><tr><td>10</td><td>9.1</td><td>1417.5</td><td>GlcNAcβ2Manα6、<br>GlcNAcβ2Manα3<br>Manβ4GlcNAcβ4GlcNAc</td><td>1.2</td><td>-</td></tr><tr><td>11<sup>d</sup></td><td>9.3</td><td>995.4</td><td>Fucα6、<br>Manβ4GlcNAcβ4GlcNAc<br>Manα3</td><td>3.9</td><td>-</td></tr><tr><td>12</td><td>9.4</td><td>833.3</td><td>Fucα6、<br>Manβ4GlcNAcβ4GlcNAc</td><td>1.3</td><td>-</td></tr><tr><td>13</td><td>10.2</td><td>995.3</td><td>Manα6、<br>Manβ4GlcNAcβ4GlcNAc</td><td>3.8</td><td>4.1</td></tr><tr><td>14</td><td>12.6</td><td>1563.5</td><td>Manα6、<br>Manβ4GlcNAcβ4GlcNAc</td><td>16.4</td><td>9.0</td></tr><tr><td>15</td><td>12.9</td><td>1360.5</td><td>Manα6、<br>Manβ4GlcNAcβ4GlcNAc<br>Manα3</td><td>3.8</td><td>5.6</td></tr><tr><td>16</td><td>15.9</td><td>1709.9</td><td>Fucα3GlcNAcβ2Manα3、<br>GlcNAcβ2Manα6<br>Manβ4GlcNAcβ4GlcNAc</td><td>2.4</td><td>3.1</td></tr><tr><td>17</td><td>16.2</td><td>1506.6</td><td>Fucα3GlcNAcβ2Manα3、<br>Manβ4GlcNAcβ4GlcNAc<br>Manα6</td><td>-</td><td>5.0</td></tr><tr><td>18</td><td>17.3</td><td>1912.8</td><td>GlcNAcβ2Manα6、<br>GlcNAcβ4、<br>Manα3、<br>Fucα3GlcNAcβ2</td><td>-</td><td>3.2</td></tr></tbody></table>
<table><caption>TABLE 2—Continued</caption><thead><tr><th rowspan="2">Peak</th><th rowspan="2">GU (ODS)<sup>a</sup></th><th rowspan="2">Observed mass value<sup>b</sup></th><th rowspan="2">Structure</th><th colspan="2">Relative quantity<sup>c</sup> (mol %)</th></tr><tr><th><i>R. varieornatus</i></th><th><i>H. exemplaris</i></th></tr></thead><tbody><tr><td>19</td><td>19.3</td><td>1855.6</td><td>Fucα3GlcNAcβ2Manα6, Manβ4GlcNAcβ4GlcNAc<br>Fucα3GlcNAcβ2Manα3</td><td>1.7</td><td>11.6</td></tr><tr><td>Others</td><td></td><td></td><td></td><td>2.6</td><td>9.3</td></tr><tr><td>Total</td><td></td><td></td><td></td><td>100</td><td>100</td></tr></tbody></table>

<sup>a</sup>GU were calculated from the elution times of the peaks obtained from the ODS column in Figure 1.

<sup>b</sup>Average mass observed as the m/z values of [M + Na]<sup>+</sup> ions for PA-oligosaccharides by MALDI-TOF-MS. The molar percentage of individual glycans within a single peak was calculated based on the peak intensity in the MALDI-TOF-MS data.

<sup>c</sup>The ratio (mol %) was calculated from the peak area in Figure 1 by comparison with the total N-glycan content derived from *R.varieornatus* and *H. exemplaris*.

<sup>d</sup>The structure did not match any entry in the GALAXY database. Structural identification was achieved through a combination of MS/MS analysis and α-mannosidase digestion (Supplemental Figs. S4 and S5).

Experimental Design and Statistical Rationale

No biological and technical replicates were performed for the gly-comics and glycoproteomics analyses due to the very limited amount of tardigrade samples available. However, the validity of the LC-MS analysis was confirmed by the consistent observation of major gly-copeptides, even when analyzing samples at one-fifth of the original volume. This study focuses on a quantitative interpretation of glyco-proteomics, demonstrating the presence of glycoproteins with the terminal Fucα1,3-GlcNAc motif.

RESULTS

Identification of N-glycans Derived From Tardigrades

N-glycans from two species of tardigrades (*R. varieornatus* and *H. exemplaris*) in an anhydrobiotic state were released via hydrazinolysis, labeled with 2-aminopyridine, and subsequently subjected to multi-dimensional HPLC profiling. Figure 1 displays the N-glycosylation HPLC-profile on an octadecyl silica (ODS) column of the pyridylaminated (PA-) glycans. The PA-oligosaccharides were identified based on the coincidence of the elution data, presented as glucose units (GU) on columns calibrated with a PA-derivatized isomalto-oligosaccharides and mass values with those in the GALAXY database (Supplemental Fig. S1 and Table 2). However, PA-glycans corresponding to fractions 16, 17, 18, and 19 did not match any of the PA-glycans previously registered in GALAXY. However, PA-glycans containing di- or tri-deoxyHex, corresponding to fractions 16, 17, 18, and 19, did not match any previously registered PA-glycans in the GALAXY database. These PA-glycans were susceptible to α-fucosidase, and their identities were subsequently registered in GALAXY. For instance, the glycosyl composition of the major α-fucosylated PA-glycan in *H. exemplaris* found in fraction 19 could be assigned as (deoxyHex)₃(Hex)₃(HexNAc)₄PA, based on its molecular mass of 1832 Da as determined by MALDI-TOF-MS. Upon α-fucosidase treatment, this glycan released three fucose residues, resulting in a biantennary oligosaccharide: GlcNAcβ1-2Manα1-6[GlcNAcβ1-2Manα1-3]Manβ1-4GlcNAcβ1-4GlcNAc-PA (code

no. 200.1 in the GALAXY database) (Supplemental Fig. S2). Its precursor structure is, therefore, a 200.1 glycan extended with two non-reducing terminal Fuc residues and one core Fuc residue. In a similar manner, the positions of non-reducing terminal Fuc residue in the PA-glycans from fractions 16, 17, and 18 were determined through HPLC mapping combined with treatments using β-N-acetylhexosaminidase and/or α-fucosidase (Table 2). To verify this assignment and further define the unusual fucosylation pattern, MALDI MS/MS sequencing was then performed on each of the permethylated derivatives of fractions 16, 17, 18, and 19 (Fig. 2).

Among the most informative fragment ions commonly afforded by the sodiated molecular ions was the Y ion at *m/z* 566, which unambiguously identified the core fucosylation. It was complemented by the B ions at *m/z* 1559, 1313, 1804, and 1733, for the molecular ions detected in fractions 16, 17, 18, and 19, respectively, fully consistent with their deduced glycosyl compositions and unambiguously localized one Fuc to the reducing end PA-tagged GlcNAc. The non-reducing terminal Fuc-HexNAc moiety was, in turn, defined by the sodiated B ion at *m/z* 456. Notably, the corresponding Fuc-HexNAc⁺ oxonium ion at *m/z* 434 was accompanied not by the loss of a MeOH moiety (–32 u) but by the elimination of a Fuc instead to yield the ion at *m/z* 228. This characteristic is strongly indicative of a Fuc 3-linked to GlcNAc (24). Further confirmation of the non-reducing terminal Fucα1,3-GlcNAc structures was provided by gas chromatography-mass spectrometry (GC-MS) linkage analysis of the total PA-glycan pool. In addition to the expected residues, including terminal Fuc (detected as 2,3,4-O-trimethyl-Fuc-ol) and 4-linked GlcNAc (2,3,6-O-trimethyl-GlcNAc-ol), 3-linked GlcNAc (2,4,6-O-trimethyl-GlcNAc-ol) and non-substituted terminal GlcNAc (2,3,4,6-O-tetramethyl-GlcNAc-ol) were also identified (Supplemental Fig. S3 and Table 1). The latter is consistent with structures deduced for fractions 16, 17, 18, and 19 (Table 2) in which one or both arms of the fucosylated trimannosyl core were extended by non-fucosylated terminal GlcNAc. In
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>FIG. 3. MS/MS fragmentation of papilin N-glycopeptides containing the Fucα1-3GlcNAc structures. Two examples of distinct glycopeptides are presented: (A) QTCEITHDQGSCRNFTVK and (B) AEPGQTVTLPCNAQGEPEPQVFWQHNNHTVR, both of which feature a HexNAc(4)Hex(3)Fuc(3) composition. EThcD fragmentation of these glycopeptides resulted in complete sequence coverage. The oxonium ion at m/z 350.15 [HexNAc(1)Fuc(1)] and fragment ions of the parent glycopeptide, excluding HexNAc(1)Fuc(2) and Fuc(2), confirm the presence of a glycan with the Fucα1,3-GlcNAc structure. According to the CFG nomenclature, blue squares, green circles, and red triangles indicate N-acetylglucosamine, mannose, and fucose, respectively.</figcaption></figure>
Total 87 N-glycoproteins

<figure><img src="image_7.png" /><figcaption>FIG. 4. A Venn diagram illustrates the identified N-glycoproteins. The overlap between N-glycoproteins expressing the non-reducing terminal Fucα1,3-GlcNAc structure and those classified as proteins with increased expression during anhydrobiosis was illustrated.</figcaption></figure>

particular, MALDI MS/MS on fractions 16 and 18, but not
fractions 17 and 19, afforded the HexNAc⁺ oxonium ion at m/z
260, whereas the FucHexNAc⁺ oxonium ion at m/z 456 was
commonly detected in all four fractions analyzed (Fig. 2). It is
evident that all four novel structures not previously registered in
GALAXY carried the uncommon terminal Fucα3GlcNAc glyco-
tope on either one or both arms of the trimannosyl core.

Identification of Glycoproteins Possessing N-Glycans With
Fucα1,3-GlcNAc Structure

To identify glycoproteins with the terminal Fucα1,3-GlcNAc
motif, we conducted LC/MSMS analysis on protein fractions
derived from *H. exemplaris* in an anhydrobiotic state. Proteins
were extracted from approximately 10,000 tardigrades, then
alkylated and digested with trypsin. The digested peptides
were loaded onto an amide-80 column to collect the
glycopeptide-enriched fraction. The glycoprotein fraction was
subsequently subjected to LC/MS analysis. Figure 3 displays
representative MS/MS spectra featuring an oxonium ion at m/
z 350.15 corresponding to the [HexNAc(1)Fuc(1)] structure and
fragment ions of the parent glycopeptide, excluding Hex-
NAc(1)Fuc(2) and Fuc(2). This indicates the presence of a
glycan with the Fucα1,3-GlcNAc antennae on glycopeptides
971 to 988 and 1579 to 1609 derived from papilin.

To ensure the most confident glycopeptide identifications,
we validated only PSMs (peptide-spectrum matches) that
corresponded to electron-transfer/higher-energy collisional
dissociation (ETHcD) spectra and met strict thresholds: a non-
negligible error probability llog Probl >2.0 and a Byonic score
>200. After applying stringent criteria, we identified a total of
8634 PSMs, leading to the discovery of 338 unique intact N-
glycopeptides derived from 87 different glycoproteins in
*H. exemplaris* (see Supplemental Table S2). Among these N-
glycoproteins, 22 exhibited the Fucα1,3-GlcNAc antenna
(refer to Fig. 4 and Table 3).

Based on the previous study that analyzed RNA sequencing
data from fully hydrated and post-desiccation samples from

*H. exemplaris* (25), a total of 15 glycoproteins showing
increased expression levels were identified and presented in
**Table 3**. Among these, eight contained the Fucα1,3-GlcNAc
antenna (Fig. 4 and Table 3).

DISCUSSION

The N-glycosylation profiles of the two tardigrade species, *R. varieornatus* and *H. exemplaris*, consist of high-mannose-type, paucimannose-type, and complex-type oligosaccharides with no detectable hybrid-type glycans. In both species, paucimannose-type oligosaccharides are among the notable components, accounting for 39% in *R. varieornatus* and 17% in *H. exemplaris*. Paucimannose-type oligosaccharides are widely found in insects and nematodes, which also belong to Ecdysozoa (26). In the tardigrade species, a significant portion of the paucimannose-type oligosaccharides undergoes fucosylation of the innermost GlcNAc residue, exclusively via α1,6-linkage. This type of core fucosylation is commonly observed in animals (27). Additionally, insects and nematodes are known to possess difucosyl core structures, where two fucose residues are attached to the 3- and 6-positions of the innermost GlcNAc (28). However, the results of HPLC and LC-MS analyses (Fig. 1 and Supplemental Fig. S6) indicate that such glycans are rarely expressed in *H. exemplari*, and represent only a minor component in *R. varieornatus*. Moreover, charged glycans were not detected as major components in our HPLC analysis. Consequently, these minor glycans were excluded from the focus of this study.

In contrast, we identified uncommon complex-type glycans
in the tardigrade species. Specifically, the complex-type oli-
gosaccharides identified in this study lack galactosylation but
undergo α1,3-fucosylation at the non-reducing terminal
GlcNAc residues.

Antenna fucosylation with an α1,3-linkage is common in
mammals, exemplified by the Lewis X antigen, but is less
prevalent in invertebrates and varies significantly among
species. In mammals, FUT9 is responsible for Lewis X for-
mation, while in *Schistosoma mansoni*, this role is fulfilled by
FUT-F. Both enzymes require Galβ1,4-GlcNAc as an acceptor
substrate (29). FucTC from the honeybee *Apis mellifera* and
the mosquito *Anopheles gambia* catalyze α1,3-fucosylation of
GalNAcβ1,4-GlcNAc as acceptor, producing an antennal
Lewis-like GalNAcβ1,4(Fucα1,3)GlcNAc moiety (30). This un-
derscores the distinctive nature of the non-reducing terminal
Fucα1,3-GlcNAc structure observed in tardigrades, which
lacks both Galβ1,4 and GalNAcβ1,4 modifications.

The antennal Fucα1,3-GlcNAc structures, devoid of Gal or
GalNAc, have been identified on N-glycans in *Taenia crassi-
cephalum* and *Manduca sexta* (31, 32). Additionally, a recent
MS analysis detected similar N-glycans with a fucosyl GlcNAc
antenna as minor glycans in some nematodes (33, 34). These
findings suggest the existence of an α1,3-fucosyltransferase
with unique substrate specificity in these organisms. A key
<table><caption>TABLE 3
List of N-glycoproteins identified in H. exemplaris</caption><thead><tr><th>Non-desiccation-inducible glycoproteins</th><th>Gene name</th><th>Protein annotation</th><th>IUPred3 score<sup>a</sup></th><th>Non-desiccation-inducible glycoproteins</th></tr></thead><tbody><tr><td rowspan="20">Glycoproteins with Fucα1, 3-GlcNAc antenna</td><td>BV898_01220</td><td>Superoxide dismutase (Cu-Zn)</td><td>0.25</td><td rowspan="20">Desiccation-inducible glycoproteins</td></tr><tr><td>BV898_03883</td><td>hypothetical protein</td><td>0.74</td></tr><tr><td>BV898_00184</td><td>PPPDE domain-containing protein</td><td>0.37</td></tr><tr><td>BV898_10055</td><td>hypothetical protein</td><td>0.01</td></tr><tr><td>BV898_08163</td><td>Chitin-binding type-2 domain-containing protein</td><td>0.00</td></tr><tr><td>BV898_01100</td><td>WAP domain-containing protein</td><td>0.84</td></tr><tr><td>BV898_05466</td><td>Papilin</td><td>0.17</td></tr><tr><td>BV898_11928</td><td>hypothetical protein</td><td>0.70</td></tr><tr><td>BV898_01512</td><td>hypothetical protein</td><td>0.04</td></tr><tr><td>BV898_01574</td><td>Transmembrane emp24 domain-containing protein 9</td><td>0.02</td></tr><tr><td>BV898_03535</td><td>FAD-linked oxidoreductase</td><td>0.01</td></tr><tr><td>BV898_04117</td><td>Vitellogenin-6</td><td>0.14</td></tr><tr><td>BV898_05246</td><td>GST N-terminal domain-containing protein</td><td>0.06</td></tr><tr><td>BV898_06105</td><td>G-protein coupled receptors family 2 profile 1 domain-containing protein</td><td>0.55</td></tr><tr><td>BV898_06922</td><td>CD109 antigen</td><td>0.11</td></tr><tr><td>BV898_06991</td><td>Neuroglian</td><td>0.32</td></tr><tr><td>BV898_08161</td><td>Chitin-binding type-2 domain-containing protein</td><td>0.00</td></tr><tr><td>BV898_08977</td><td>Pancreatic lipase-related protein 2</td><td>0.02</td></tr><tr><td>BV898_09795</td><td>L-sorbosone dehydrogenase</td><td>0.01</td></tr><tr><td>BV898_11190</td><td>Laminin subunit alpha</td><td>0.11</td></tr><tr><td>BV898_13158</td><td>Chitin-binding type-2 domain-containing protein</td><td>0.12</td></tr><tr><td>BV898_13788</td><td>Nidogen-1</td><td>0.17</td></tr><tr><td rowspan="19">Glycoproteins without Fucα1, 3-GlcNAc antenna</td><td>BV898_09026</td><td>Secreted protein</td><td>0.00</td><td rowspan="19">Desiccation-inducible glycoproteins</td></tr><tr><td>BV898_12027</td><td>hypothetical protein</td><td>0.38</td></tr><tr><td>BV898_06777</td><td>von Willebrand factor A domain-containing protein</td><td>0.14</td></tr><tr><td>BV898_06906</td><td>MATH domain-containing protein</td><td>0.00</td></tr><tr><td>BV898_06776</td><td>von Willebrand factor A domain-containing protein</td><td>0.25</td></tr><tr><td>BV898_03546</td><td>hypothetical protein</td><td>0.10</td></tr><tr><td>BV898_00260</td><td>Sarcalumenin</td><td>0.26</td></tr><tr><td>BV898_00022</td><td>hypothetical protein</td><td>0.11</td></tr><tr><td>BV898_00065</td><td>Endoplasmic reticulum-Golgi intermediate compartment protein 1</td><td>0.04</td></tr><tr><td>BV898_00095</td><td>hypothetical protein</td><td>0.50</td></tr><tr><td>BV898_00099</td><td>putative Apolipoprotein B-100</td><td>0.18</td></tr><tr><td>BV898_00100</td><td>Ectonucleoside triphosphate diphosphohydrolase 2</td><td>0.04</td></tr><tr><td>BV898_00577</td><td>putative Beta-1,3-galactosyltransferase 5</td><td>0.01</td></tr><tr><td>BV898_00612</td><td>hypothetical protein</td><td>0.18</td></tr><tr><td>BV898_00911</td><td>hypothetical protein</td><td>0.19</td></tr><tr><td>BV898_02207</td><td>putative Apolipoprotein D</td><td>0.01</td></tr><tr><td>BV898_02239</td><td>Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1</td><td>0.63</td></tr><tr><td>BV898_02454</td><td>Mitochondrial ribonuclease P protein 3</td><td>0.03</td></tr><tr><td>BV898_02736</td><td>hypothetical protein</td><td>0.00</td></tr><tr><td>BV898_02837</td><td>Lysosomal alpha-glucosidase</td><td>0.02</td></tr><tr><td>BV898_02893</td><td>putative Pancreatic triacylglycerol lipase</td><td>0.00</td></tr><tr><td>BV898_03230</td><td>DBH-like monooxygenase protein 1-like protein</td><td>0.06</td></tr></tbody></table>
<table><caption>TABLE 3—Continued</caption><thead><tr><th>Non-desiccation-inducible glycoproteins</th><th>Gene name</th><th>Protein annotation</th><th>IUPred3 score<sup>a</sup></th><th>Non-desiccation-inducible glycoproteins</th></tr></thead><tbody><tr><td></td><td>BV898_03257</td><td>putative Leucine-rich repeat-containing protein 15</td><td>0.00</td><td></td></tr><tr><td></td><td>BV898_03417</td><td>hypothetical protein</td><td>0.15</td><td></td></tr><tr><td></td><td>BV898_03425</td><td>Sodium/potassium-transporting ATPase subunit beta-2</td><td>0.07</td><td></td></tr><tr><td></td><td>BV898_03478</td><td>hypothetical protein</td><td>0.00</td><td></td></tr><tr><td></td><td>BV898_03587</td><td>hypothetical protein</td><td>0.08</td><td></td></tr><tr><td></td><td>BV898_03819</td><td>putative Ras association domain-containing protein 1</td><td>0.12</td><td></td></tr><tr><td></td><td>BV898_03853</td><td>hypothetical protein</td><td>0.01</td><td></td></tr><tr><td></td><td>BV898_04264</td><td>Cathepsin B</td><td>0.04</td><td></td></tr><tr><td></td><td>BV898_04276</td><td>Laminin subunit gamma-1</td><td>0.09</td><td></td></tr><tr><td></td><td>BV898_04951</td><td>Epoxide hydrolase 1</td><td>0.00</td><td></td></tr><tr><td></td><td>BV898_05042</td><td>hypothetical protein</td><td>0.09</td><td></td></tr><tr><td></td><td>BV898_05629</td><td>hypothetical protein</td><td>0.12</td><td></td></tr><tr><td></td><td>BV898_05653</td><td>hypothetical protein</td><td>0.08</td><td></td></tr><tr><td></td><td>BV898_06078</td><td>putative Zinc metalloproteinase nas-13</td><td>0.12</td><td></td></tr><tr><td></td><td>BV898_06174</td><td>Serpin B9</td><td>0.00</td><td></td></tr><tr><td></td><td>BV898_06377</td><td>hypothetical protein</td><td>0.09</td><td></td></tr><tr><td></td><td>BV898_06476</td><td>putative Testicular acid phosphatase-like protein</td><td>0.18</td><td></td></tr><tr><td></td><td>BV898_06985</td><td>hypothetical protein</td><td>0.69</td><td></td></tr><tr><td></td><td>BV898_07240</td><td>hypothetical protein</td><td>0.03</td><td></td></tr><tr><td></td><td>BV898_07373</td><td>putative CD63 antigen</td><td>0.00</td><td></td></tr><tr><td></td><td>BV898_07785</td><td>hypothetical protein</td><td>0.04</td><td></td></tr><tr><td></td><td>BV898_08327</td><td>putative Insulin-like growth factor-binding protein complex acid labile subunit</td><td>0.00</td><td></td></tr><tr><td></td><td>BV898_08330</td><td>Vitellogenin-6</td><td>0.09</td><td></td></tr><tr><td></td><td>BV898_08568</td><td>Alkyldihydroxyacetonephosphate synthase, peroxisomal</td><td>0.05</td><td></td></tr><tr><td></td><td>BV898_08580</td><td>Arylsulfatase J</td><td>0.17</td><td></td></tr><tr><td></td><td>BV898_08836</td><td>putative Translocon-associated protein subunit alpha</td><td>0.14</td><td></td></tr><tr><td></td><td>BV898_09465</td><td>putative Translocon-associated protein subunit beta</td><td>0.06</td><td></td></tr><tr><td></td><td>BV898_09643</td><td>hypothetical protein</td><td>0.00</td><td></td></tr><tr><td></td><td>BV898_09705</td><td>Cullin-4A</td><td>0.03</td><td></td></tr><tr><td></td><td>BV898_10022</td><td>hypothetical protein</td><td>0.08</td><td></td></tr><tr><td></td><td>BV898_10209</td><td>hypothetical protein</td><td>0.13</td><td></td></tr><tr><td></td><td>BV898_10240</td><td>hypothetical protein</td><td>0.24</td><td></td></tr><tr><td></td><td>BV898_10963</td><td>hypothetical protein</td><td>0.21</td><td></td></tr><tr><td></td><td>BV898_11681</td><td>Protein mesh</td><td>0.15</td><td></td></tr><tr><td></td><td>BV898_11843</td><td>Dynein heavy chain 7, axonemal</td><td>0.04</td><td></td></tr><tr><td></td><td>BV898_12405</td><td>putative Carbonic anhydrase 14</td><td>0.14</td><td></td></tr><tr><td></td><td>BV898_12554</td><td>Integrin alpha-PS2</td><td>0.09</td><td></td></tr><tr><td></td><td>BV898_12667</td><td>Chitinase-3-like protein 1</td><td>0.00</td><td></td></tr><tr><td></td><td>BV898_12870</td><td>hypothetical protein</td><td>0.19</td><td></td></tr><tr><td></td><td>BV898_13159</td><td>hypothetical protein</td><td>0.00</td><td></td></tr><tr><td></td><td>BV898_13336</td><td>hypothetical protein</td><td>0.22</td><td></td></tr><tr><td></td><td>BV898_13836</td><td>hypothetical protein</td><td>0.46</td><td></td></tr><tr><td></td><td>BV898_14266</td><td>Multidrug resistance-associated protein 1</td><td>0.10</td><td></td></tr></tbody></table>

<footnote><sup>a</sup>IUPred3 score of 0.5 or more are shown in red. IUPred3 is a tool that identifies intrinsically disordered protein regions using a biophysics-based model, providing a score between 0 and one for each residue to indicate the likelihood of that residue being part of a disordered region (35).</footnote>
finding of this study is that the *N*-glycans with this distinctive structure are prevalent in *H. exemplaris*.

This distinctive glycosylation is more prevalent in *H. exemplaris*. Among the 87 *N*-glycoproteins identified from *H. exemplaris*, 22 glycoproteins expressed the Fucα1,3-GlcNAc structure (Fig. 4). Notably, eight of these glycoproteins were induced during anhydrobiosis, including Cu/Zn-superoxide dismutase (SOD) and papilin (Table 3).

The SOD enzyme is an antioxidant that is secreted extracellularly and located between tardigrade cells, protecting the organism from oxidative stress. Conversely, papilin is thought to promote the reconstruction and remodeling of extracellular matrix by regulating the activity of metalloproteases. SOD has a single *N*-linked glycosylation site, to which a biantennary complex-type glycan with or without the non-reducing terminal Fucα1,3-GlcNAc structure was attached. For papilin, out of the 21 potential *N*-linked glycosylation sites, glycan information was obtained for two sites. All the detected papilin glycopeptides possessed *N*-glycans containing the Fucα1,3-GlcNAc antenna. The functions and localization of these protective proteins may be influenced by their *N*-glycosylation patterns.

Among the eight glycoproteins with desiccation-inducible properties that contain the Fucα1,3-GlcNAc antenna, three—annotated as BV898_03883, BV898_01100, and BV898_11928—exhibited high IUPred3 scores. This tool identifies intrinsically disordered protein regions (IDRs) using a biophysics-based model (35). Notably, BV898_01100 and BV898_11928 have been confirmed to display *N*-glycans featuring the Fucα1,3-GlcNAc antenna at regions predicted to be IDRs. In tardigrades, several proteins with IDRs are supposed to play roles in the cryptobiosis mechanism. Consequently, these glycoproteins may also be critical during desiccation, potentially leveraging their distinctive *N*-glycans to recruit proteins with lectin activity, among other functions, thereby significantly contributing to this process.

Tardigrade genomic analyses identified several genes encoding homologs of FUT9 (Uniport entry name: FUT9_HUMAN) and FucTC (Uniport entry name: Q05GU1_APICA) (Supplemental Tables S3 and S4). Recombinant expression of these candidate enzymes, along with recently developed techniques for gene expression and suppression in tardigrades, will enable elucidation of the molecular mechanisms underlying their distinctive glycosylation. While our study did not involve glycoproteomic analysis of active tardigrades, and therefore whether the Fucα1,3-GlcNAc-containing glycans themselves are upregulated during desiccation remains to be elucidated, this work provides a crucial foundation for future investigations into the role of glycans in tardigrade anhydrobiosis.

DATA AVAILABILITY

The data presented in this study are available upon request from the corresponding authors. The LC data, along with the subsequent MALDI-TOF-MS data, and the MS-based

glycoproteomics data have been deposited in GlycoPOST (Accession No. GPST000534) and JPOST (Accession No. JPST003355), respectively.

**Supplemental Data**—This article contains Supplemental Data.

**Acknowledgments**—We are grateful to the Analytical Center, Meijo University for the MALDI-TOF/MS measurements of oligosaccharides. The *C. vulgaris* used to feed the tardigrades was provided courtesy of Chlorella Industry. The authors thank Naoko Ishii for technical assistance.

**Author Contributions**—K. T., K. K., Y. G., S. K., S. S., N. Y., H. Y., K. A., S. G., J. F., and T. S. investigation; M. Y., K. K., Y. G., N. Y., and K. A. writing–review & editing; M. Y. and K. K. project administration; K. K. and Y. G. resources; Y. G., K. K., and H. Y. conceptualization; K. K. and H. Y. writing–original draft; K. K. and K. A. supervision; K. K., H. Y., and K. A. funding acquisition; H. Y.: visualization; H. Y. validation; H. Y. data curation.

**Funding and Additional Information**—This work was supported in part by the Human Glycome Atlas Project (HGA), by the J-GlycoNet cooperative network MEXT, as a Joint Usage/Research Center, by MEXT/JSPS KAKENHI (Grant Number JP24H00599 to K.K., 21H05279 to K.A.), JST FOREST Program (Grant Number JPMJFR2255 to H.Y.), by JST CREST (Grant Number JPMJCR21E3 to K.K.), and funds from Yamagata Prefectural Government and Tsuruoka City. This work was also supported by the Joint Research of the Exploratory Research Center on Life and Living Systems (ExCELLS) (ExCELLS program No. 18–207, 19–208, and 19–501 to K.A.), by ExCELLS Project Research (ExCELLS program No. 22EXC601 and 23EXC601). This work was the result of using research equipment shared in MEXT Project for promoting public utilization of advanced research infrastructure (Program for supporting construction of core facilities, JPMXS0441500024. We thank the Academia Sinica Common Mass Spectrometry Facilities for Proteomics and Protein Modification Analysis for MALDI-MS/MS sequencing data collection.

**Conflicts of Interests**—The authors declare that they have no conflicts of interest with the contents of this article.

**Abbreviation**—The abbreviations used are: CAHS, cytoplasmic abundant heat soluble; CID, collision-induced dissociation; EThcD, electron-transfer/higher-energy collisional dissociation; Fuc, fucose; GC-EI-MS, Gas chromatography-electron impact MS; GlcNAc, *N*-acetylglucosamine; HCD, higher-energy collisional dissociation; Hex, hexose; HexNAc, *N*-acetylhexosamine; IDR, intrinsically disordered protein region; LEAM, mitochondrial late embryogenesis abundant;
MAHS, mitochondrial abundant heat soluble: Man, mannose; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NMR, Nuclear Magnetic Resonance; ODS, octadecyl silica; PA, pyridylaminated; PSM, peptide-spectrum match; SAHS, secretory abundant heat soluble; SOD, Cu/Zn-superoxide dismutase.

Received October 18, 2024, and in revised form, April 15, 2025
Published, MCPro Papers in Press, April 28, 2025, https://doi.org/
10.1016/j.mcpro.2025.100979

REFERENCES

1. Giribet, G., and Edgecombe, G. D. (2019) The phylogeny and evolutionary history of arthropods. *Curr. Biol.* **29**, R592–R602

2. Degma, P., Bertolani, R., and Guidetti, R. (2019) Actual Checklist of Tardi-
grada Species. https://hdl.handle.net/11380/1178608 (Accessed 16 May
2025)

3. Hengherr, S., Worland, M. R., Reuner, A., Brümmer, F., and Schill, R. O. (2009) High-temperature tolerance in anhydrobiotic tardigrades is limited by glass transition. *Physiol. Biochem. Zool.* **82**, 749–755

4. Horikawa, D. D., Sakashita, T., Katagiri, C., Watanabe, M., Kikawada, T., Nakahara, Y., et al. (2006) Radiation tolerance in the tardigrade Milnesium tardigradum. *Int. J. Radiat. Biol.* **82**, 843–848

5. Jönsson, K. I., Rabbow, E., Schill, R. O., Harms-Ringdahl, M., and Rettberg, P. (2008) Tardigrades survive exposure to space in low Earth orbit. *Curr. Biol.* **18**, R729–R731

6. Schill, R. O., Mali, B., Dandekar, T., Schnölzer, M., Reuter, D., and Frohme, M. (2009) Molecular mechanisms of tolerance in tardigrades: new perspectives for preservation and stabilization of biological material. *Bio-technol. Adv.* **27**, 348–352

7. Arakawa, K. (2022) Examples of extreme survival: tardigrade genomics and molecular anhydrobiology. *Annu. Rev. Anim. Biosci.* **10**, 17–37

8. Horikawa, D. D., Kunieda, T., Abe, W., Watanabe, M., Nakahara, Y., Yukuhiro, F., et al. (2008) Establishment of a rearing system of the extremotolerant tardigrade Ramazzottius varieornatus: a new model animal for astrobiology. *Astrobiology* **8**, 549–556

9. Gabriel, W. N., McNuff, R., Patel, S. K., Gregory, T. R., Jeck, W. R., Jones, C. D., et al. (2007) The tardigrade Hypsibius dujardini, a new model for studying the evolution of development. *Dev. Biol.* **312**, 545–559

10. Yagi-Utsumi, M., Aoki, K., Watanabe, H., Song, C., Nishimura, S., Satoh, T., et al. (2021) Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade. *Sci. Rep.* **11**, 21328

11. Miyazawa, K., Itoh, S. G., Watanabe, H., Uchihashi, T., Yanaka, S., Yagi-
Utsumi, M., et al. (2021) Tardigrade secretory-abundant heat-soluble
protein has a flexible β-barrel structure in solution and keeps this struc-
ture in dehydration. *J. Phys. Chem. B* **125**, 9145–9154

12. Yamaguchi, A., Tanaka, S., Yamaguchi, S., Kuwahara, H., Takamura, C., Imajoh-Ohmi, S., et al. (2012) Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade. *PLoS One* **7**, e44209

13. Tanaka, S., Tanaka, J., Miwa, Y., Horikawa, D. D., Katayama, T., Arakawa, K., et al. (2015) Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells. *PLoS One* **10**, e0118272

14. Arakawa, K., Yoshida, Y., and Tomita, M. (2016) Genome sequencing of a single tardigrade Hypsibius dujardini individual. *Sci. Data* **3**, 160063

15. Yagi, H., Yamamoto, M., Yu, S.-Y., Takahashi, N., Khoo, K.-H., Lee, Y. C., et al. (2010) N-Glycosylation profiling of turtle egg yolk: expression of galabiose structure. *Carbohydr. Res.* **345**, 442–448

16. Yagi, H., Yasukawa, N., Yu, S.-Y., Guo, C.-T., Takahashi, N., Takahashi, T., et al. (2008) The expression of sialylated high-antennary N-glycans in edible bird's nest. *Carbohydr. Res.* **343**, 1373–1377

17. Takahashi, N., Yagi, H., and Kato, K. (2008). In: Kamerling, J., et al., eds., *2. Comprehensive Glycoscience*, Elsevier, Oxford: 283–301

18. Yagi, H., Amagasa, E., Shiota, M., Yamada, I., Aoki-Kinoshita, K. F., and Kato, K. (2022) GALAXY ver3: updated web application for glycosylation profiling based on 3D HPLC map. *Glycobiology* **32**, 646–650

19. Takahashi, N., and Kato, K. (2003) GALAXY(glycoanalysis by the three axes of MS and chromatography): a web application that assists structural analyses of N-glycans. *Trends Glycosci. Glycotechnol.* **15**, 235–251

20. Anumula, K. R., and Taylor, P. B. (1992) A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates. *Anal. Biochem.* **203**, 101–108

21. Sriwilaijaroen, N., Kondo, S., Yagi, H., Wilairat, P., Hiramatsu, H., Ito, M., et al. (2009) Analysis of N-glycans in embryonated chicken egg chorio-allantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses. *Glycoconj. J.* **26**, 433–443

22. Togayachi, A., Tomioka, A., Fujita, M., Sukegawa, M., Noro, E., Takakura, D., et al. (2018) Identification of poly-N-acetyllactosamine-carrying glycoproteins from HL-60 human promyelocytic leukemia cells using a site-specific glycome analysis method, Glyco-RIDGE. *J. Am. Soc. Mass Spectrom.* **29**, 1138–1152

23. Nakagawa, S., Imachi, H., Shimamura, S., Yanaka, S., Yagi, H., Yagi-
Utsumi, M., et al. (2024) Characterization of protein glycosylation in an
Asgard archaeon. *BBA Adv.* **6**, 100118

24. Dell, A., Reason, A. J., Khoo, K. H., Panico, M., McDowell, R. A., and Morris, H. R. (1994) Mass spectrometry of carbohydrate-containing bio-polymers. *Methods Enzymol.* **230**, 108–132

25. Yoshida, Y., Koutsovoulos, G., Laetsch, D. R., Stevens, L., Kumar, S., Horikawa, D. D., et al. (2017) Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus. *PLoS Biol.* **15**, e2002266

26. Paschinger, K., and Wilson, I. B. H. (2019) Comparisons of N-glycans across invertebrate phyla. *Parasitology* **146**, 1733–1742

27. Yang, Q., and De Schutter, K. (2023) Insights into protein fucosylation in insects. *Entomol. Gen.* **43**, 911–925

28. Schiller, B., Hykollari, A., Yan, S., Paschinger, K., and Wilson, I. B. H. (2012) Complicated N-linked glycans in simple organisms. *Biol. Chem.* **393**, 661–673

29. Mickum, M. L., Rojsajjakul, T., Yu, Y., and Cummings, R. D. (2016) Schis-
tosoma mansoni α1,3-fucosyltransferase-F generates the Lewis X anti-
gen. *Glycobiology* **26**, 270–285

30. Kurz, S., King, J. G., Dinglasan, R. R., Paschinger, K., and Wilson, I. B. H. (2016) The fucomic potential of mosquitoes: fucosylated N-glycan epitopes and their cognate fucosyltransferases. *Insect Biochem. Mol. Biol.* **68**, 52–63

31. Stephens, E., Sugars, J., Maslen, S. L., Williams, D. H., Packman, L. C., and Ellar, D. J. (2004) The N-linked oligosaccharides of aminopeptidase N from Manduca sexta: site localization and identification of novel N-glycan structures: site localization and identification of novel N-glycan structures. *Eur. J. Biochem.* **271**, 4241–4258

32. Lee, J. J., Dissanayake, S., Panico, M., Morris, H. R., Dell, A., and Haslam, S. M. (2005) Mass spectrometric characterization of Taenia crassiceps metacestode N-glycans. *Mol. Biochem. Parasitol.* **143**, 245–249

33. Petralia, L. M. C., van Diepen, A., Lokker, L. A., Nguyen, D. L., Sartono, E., Khatri, V., et al. (2022) Mass spectrometric and glycan microarray-based characterization of the filarial nematode Brugia malayi glycome reveals anionic and zwitterionic glycan antigens. *Mol. Cell. Proteomics* **21**, 100201

34. Paschinger, K., Wöls, F., Yan, S., Jin, C., Vanbeselaere, J., Dutkiewicz, Z., et al. (2023) N-glycan antennal modifications are altered in Caeno-rhabditis elegans lacking the HEX-4 N-acetylgalactosamine-specific hexosaminidase. *J. Biol. Chem.* **299**, 103053

35. Erdős, G., Pajkos, M., and Dosztányi, Z. (2021) IUPred3: prediction of protein disorder enhanced with unambiguous experimental annotation and visualization of evolutionary conservation. *Nucleic Acids Res* **49**, W297–W303

36. Yu, S.-Y., Wu, S.-W., and Khoo, K.-H. (2006) Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans. *Glycoconj. J.* **23**, 355–369

37. Neelamegham, S., Aoki-Kinoshita, K., Bolton, E., Frank, M., Lisacek, F., Lütteke, T., et al. (2019) Updates to the symbol nomenclature for glycans guidelines. *Glycobiology* **29**, 620–624