Peer review of: Prominent members of the human gut microbiota express endo-acting O-glycanases to (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The colonic mucus barrier separates the human gut microbiome (both commensals and pathogens) 
from the epithelium and plays a vital role as primary defense against the enteric pathogens. 
Recent literature underscores the importance of this barrier for the host immune system and thus, 
the crosstalk between the microbiome and the mucus barrier is a decisive event in health and 
disease. The microbiome is armored with an array of enzymes that specifically cleave variety of 
linkages in the host mucus. So far, this model of microbial metabolism of host-secreted mucus 
involves only exo-acting enzymes, meaning that the enzymes are known to act on the linkages 
connecting the terminal sugars of the mucin glycoprotein. 

Here, Crouch et al. report discovery of endo-acting O-glycanases in select human gut bacterial 
species belonging to two major phyla Bacteroidetes (3 species; Bacteroidetes thetaiotaomicron, B. 
fragilis and B. caccae) and Verrucomicrobia (A. muciniphila). First, the authors use data from 
previously published studies from two other labs that reported higher expression of certain 
transcripts/proteins belonging to the glycoside hydrolase (GH) family 16. Next, they used mass 
spectrometry to demonstrate that cloned protein products of these transcripts act on mucus 
polysaccharides (porcine gastric mucus) in an endo-acting manner. The authors then performed a 
detailed biochemical characterization (including analyese of the crystal structures) of these 
proteins to further support the endo-acting nature of these enzymes on the colonic mucus. Finally, 
using limited samples from human patients (ulcerative colitis, necrotising enterocolitis) and 
human-derived colorectal cancer cell lines, the authors show that the endo-acting ability of these 
enzymes also extends to human-derived mucins. 

The study is certainly informative and the discovery of endo-acting glycanases sheds important 
light on the mechanisms behind how the gut microbiome interacts with the mucus barrier. One 
major issue is that the patient sample size is small. Given this, the patient data cannot be 
conclusive – I suggest that either the authors increase their cohort size or exclude the patient data 
from the current manuscript or do not make definitive conclusions from the patient data. To 
improve quality of the manuscript, I have several comments to re-formulate the text. 

General comments 

Identification of GH16 enzymes expressed during growth on mucin: The authors have used data 
from three previous studies (Martens, Cell Host Microbe, 2008; Desai, Cell, 2016; and Ottmann, 
Applied and Environmental Microbiology, 2017). It might be useful to explain the rationale behind 
shortlisitng some specific transcripts and not others and also why only these four specific species 
were chosen. Fig. S21 shows that there are also other Bacteroidetes strains that can grown on 
mucus glycans. Although Fig. 1 is useful to understand how bacteria metabolize the mucus 
polysaccharides, as this figure does not have new data, it might be good to have it as a 
supplemental figure. Authors can use previously published reviews (e.g., Martens, Nature Reviews 
Microbiology, 2018) to highlight these biochemical aspects related to mucus degradation. Instead, 
it might be useful to provide some details of the localization of the identified GH16 enzymes within 
the bacterial cell wall. This should be generally explored in more detail to offer or characterize a 
mechanism. How mucin O-glycans are degraded by the endo-hydrolyases (first, OM-attached 
enzyme X cleaves sugar residue Y, then transport into perisplasmic space, then enzyme Z 
catalyzes; something like this). Amuc_2108 seems to be located in the OM, while other GH16 
enzymes (Amuc_0724 and Amuc_0875 seem to be either in the periplasmic space or in the IM?). 

GH16 enzymes are endo-acting mucinases: 
Fig. 2a: Generation of recombinant forms of the 9 identified GH16 enzymes from BT, BF, BC and 
AM. They test their activity to hydrolyze either porcine SI mucin, procine gastric mucin type PGM II 
and procine gastric mucin type PGM III. 

Comment: Do the recombinant forms of the enzymes still contain their lipid anchors? They refer to 
Fig. S8. What do they show there? Is this a thin layer chromatography?? What is the control? 

Figure 2b: Then, they chose the products of BF4058 of SI mucin for further characterization. 
Comment: why this enzyme and why SI mucin? 

Products were treated with an exo-acting hydrolyse. 
Comment: It’s unclear why they do this experiment and what is the conclusion. Everything they 
“conclude” is “likely” or “possibly”. 
Furthermore, they have already investigated the structure via MS/MS… 
Fig. 2B: Please clarify what is meant by the red star? 

Next, they tested the capability of the 9 enzymes to degrade keratan sulfate, which is structurally 
similar to mucin O-glycans. They detect that the 9 enzymes are active against keratan sulfates and 
that the enzymes can tolerate “a significant number of sulfates”. 
Comment: Why is this experiment done? What about the sulfates in the mucins they used in 
previous experiments? 

Next, they used a range of defined oligosaccharides to assess the substrate specificity of the 9 
enzymes 
Comment: Data and text are sound. However, a combining or conluding main figure would be nice 
for the main manuscript 

Next, they tested the hydrolysis activity of the 9 enzmyes towards previously published validated 
GH16 substrates 
Comment: They could not really detect any hydrolysis of these substrates (besides some 
exeptions, which are disccused in the SM) 
But: what is the conclusion? GH16 enzymes with specificity for mucins? What’s the difference and 
the final conclusion? 

Overall, this paragraph is not really clear and comprehensive. The data of Fig. 2A are nice. 
However, it should be made clear what the purpose of Fig. 2B is. Why this is done and what are 
the conclusions of the “story”. The text should mention that the red asterisks refer to the 
disappeared peaks. 

Crystal structures: 
Comment: Perhaps here they should clearly mention that this is done to elucidate the structural 
basis for mucin-specificity? 

They analyzed the apo-structures of BC2680, BC2680E143Q, BC3717 and AM0724. 
Comment: Why were they chosen? Why the mutant? 

Additionally, BF4060 and BC2680E143Q together with a trisaccharide. 
Comment: What is the purpose of this? Why now adding BF4060? Why not comparing BC2680 
with its mutant? Is this a catalytic mutant? Why not only perform co-crystallization of the holo-
enzyme together with the substrate? Where does the trisaccharide come from? Was it added to the 
enzyme in its trisaccharide form or is it the product of an enzymatic reaction by these enzymes? 

Activity against human mucins: 
Comment: It should be mentioned in the text how the “human mucin samples” were generated 
and processed. 

They incubated the human mucin samples with AM0724 (why only this one?) and sialidase. 
Products were then labeled and identified. 
They conclude that “enzymes could be used to characterise the glycan structures present in 

mucins from different disease states” 
Comment: This conclusion is an overinterpretation and not supported by the available data. Is 
there a real difference between different disease states? There are no samples from healthy 
controls and only 2 samples from UC patients. The sample choice (uninflamed ileum and inflamed 
colon from Patient 1; inflamed ileum, uninflamed ileum and uninflamed colon) seems to be very 
random (and n=1 for each condition). The overall conclusion “showed different patterns of 
oligosaccharides released depending on the disease and tissue type” is not supported by the data. 
There are too many variables (individual, disease type, inflammation state, tissue sample, …) and 
such a conclusion cannot be made by analyzing samples from only 1-2 individuals with a random 
sample selection. I am not certain about showing these data with definitive conclusions. I suggest 
to beef up the cohort size or tone down the conclusion based on the human data (including data 
on the CRC cell lines). 

Reviewer #2 (Remarks to the Author): 

This manuscript by Crouch et al. on the ability of O-glycanases from human gut microbiota 
members to digest mucin O-glycans via an endo-acting mechanism is well-supported by analytical 
and X-ray crystallographic studies. Some additional experiments and discussion are suggested to 
enhance the impact of this manuscript. 

Major claims: (1) Gut bacteria access mucin O-glycans in the human intestine as a nutrient source. 
(2) Instead of acting as exoglycosidases, the enzymes secreted by these gut bacteria act via an 
endoglycosidase mechanism. (3) Understanding this mechanism is key to understanding human 
gut microbiota interactions. (4) Recombinant O-glycanases can be useful tools to explore changes 
in the O-glycome of IBD and colorectal cancer. 

Evaluation: There is significant interest in the human gut microbiome, and this manuscript 
describes detailed biochemical and structural evidence that glycosidases of the GH16 family from 
Bacteroides and Akkermansia act by digesting mucin glycans from porcine intestine via an endo 
mechanism instead of exo. Biochemical evidence in the form of glycan LCMS is adequate, although 
the authors are suggested to further simplify Figure 2 for the non-glycoscientist. In particular, it is 
unclear why it is significant to group the enzymes based on products of more or less than four 
sugars (seems arbitrary). It would also be helpful to compare the structures of human versus 
porcine intestinal mucin to provide relevance to the study, as well as gain a biochemical 
understanding of the expression levels of the enzymes describes in the bacteria specified (only 
previously reported transcriptomic dataset is referenced). The cloning and expression of the 
recombinant proteins should also be described (right now the reader is referred to reference 25). 
Enzymes used in this study were produced in an E. coli mutant, but how (if any at all) abundantly 
are these enzymes expressed, and how abundant are the requisite sialidases? Are these enzymes 
also active on N-glycans with extended poly-LacNAc chains? N-glycans are also prominent 
members of the gut glycome. Is there specificity at the protein level as to which mucin 
glycoproteins are degraded by these enzymes (doesn't seem to, but explicit mention of this point 
would be helpful). Can the SusC/D outer membrane complex be genetically manipulated to show 
that there is indeed uptake of the broken down glycan byproducts that could then be used for 
nutrition? More sophisticated experiments that more closely mimic the nature of the human gut 
microbiome would greatly enhance this manuscript. Is mucosal viscosity a factor in the activity? 
What about pH? The pH in the small intestine is ~ pH 6 and ~7.4 in the terminal ileum. 

Overall, these findings are of specific interest to the glycoscience-gut microbiome community, and 
the conclusions are original, but would be further strengthened by the suggested experiments. 
There is additional impact in adding information to carbohydrate-active enzymes. While the 
authors' experimental conclusions are well supported, subjectively, I find a large disconnect in this 
small detail (exo vs endo mechanism) and it being incredibly significant and key to understanding 

gut-microbe interactions. It is not clear that this is a highly debated topic that is of general 
widespread interest to the wider community and that it is a key step to influence thinking in the 
field (although a better discussion of these specific topics could influence my evaluation). In my 
opinion, there is greater impact on the utility of these enzymes as tools to determine changes in 
patient samples, but there is not adequate data here for comparison (healthy samples are limited). 
Again, figure 4 can be better represented as digested data with bar graphs and statistical analysis 
instead of a series of chromatograms. 

Statistical analysis: Although the chromatograms used for glycan analysis are included and the 
description of methods used to generate them are satisfactory, there are limited mentions of any 
statistical analysis used. Instead of the chromatograms, bar graphs depicting the major 
conclusions replete with statistical measures of reproducibility are suggested. 

Reviewer #3 (Remarks to the Author): 

This manuscript that reports a substantial volume of work on the identification and detailed 
characterization of the first endo-acting mucinases from a number of species of prominent human 
gut bacteria spanning two distinct phyla. Mucin samples from human were also shown to be 
degraded by one of the characterized enzymes. These endo-mucinases represent the first reported 
of such specificity in the large and well-studied glycoside hydrolase family 16, making this work of 
great interest to the carbohydrate enzyme community as well as to the gut microbiota community. 

Regrettably, the presentation of the manuscript leaves much to be desired, and in many instances 
a clear lack of attention to detail is evident. It would seem that that the numerous co-authors have 
not carefully proofread the manuscript, which is burdensome to reviewers and regrettable 
considering the quality of the journal the authors are targeting. It is incumbent upon the 
corresponding authors to ensure that this is done properly. 

The following comments must be addressed. These are presented generally in order of appearance 
in the documents. 

Main text 
Typographical/grammatical/sentence-structure problems must be addressed, for example on lines: 
57, 58, 106, 116, 182, 278, 321, 354, 372, 378, 441, 445, 465, 499, 520. Note that this does not 
comprise an exhaustive list and the manuscript must be checked carefully throughout. 

The sections titled “The GH16 enzymes are endo-acting mucinases” and “Crystal structures of O-
glycan active GH16 family members” are big sections that present the bulk of the data; their 
content should be divided into smaller subsections with informative headers to improve readability. 

It would be more logical for the section titled “Phylogenetic analysis of the GH16 O-glycanases” to 
be moved up, directly after the section “Identification of GH16 enzymes expressed during growth 
on mucin”. Also, this way it is consistent with the figure order and supplementary discussion order. 
Content that is best presented after the enzymology and structural biology sections can go in the 
discussion. 

For the section titled “The GH16 O-glycanases are active against human mucins from healthy and 
diseased samples”, more interpretation of the results is required. The first paragraph describes the 
sample source, the second paragraph is discussion material (should really be moved to the 
discussion section), and only the third paragraph (lines 327-333) describes the results. Maybe 
elaborate a bit on the pattern of oligosaccharides released as a function of disease state. In light of 
the possibility of use as a biomarker, are there perhaps any particular unique or uniquely enriched 
oligosaccharides corresponding to a disease? 

Line 95: Define “Sus” and explain. 

Line 109-112: with regard to ref. 29, it would seem that the linkage specificity of the enzymes in 
the present study is not entirely unprecedented/surprising. Here, and in the corresponding 
structural biology sections, the structural similarities and differences with regard to carbohydrate 
recognition should be explicitly described, including through the use of structural superpositions. 

Line 114: What does “sequence diverse” mean in this context? Presumably, a subfamily would 
have comparatively low diversity compared with the larger GH16 family. 

Line 128: PGM = porcine gastric mucin. 

Line 144: Please elaborate on the structural basis for this specificity, especially with regard to the 
cleft-like active-site of these enzymes (see also comment on Lines 109-112 above). 

Line 154: Where is the data that shows this? I think it is supposed to be in Supplemental Figure 13 
but is missing (see below). 

Line 155: Was there a particular reason for the selection of BT4058 for detailed product analysis? 

Line 212-217: There are six structures described here but only stats for four of the structures in 
sup table 5. 

Line 217: Sup figs 17-19 not 15-17. 

Line 214-218: Privateer [Nature Structural & Molecular Biology volume 22, pages 833–834 (2015)] 
must be used to validate these sugar models and the resulting output reported in a supplemental 
table. (See also identical comment in SI section below). 

Line 276: Meaning unclear (misplaced modifier). 

Line 271: Sup fig 16 not 18. 

Line 281: Run-on sentence/poor grammar. 

Line 327: Was there a particular reason for the selection of Amuc_0724 for assays with human 
mucin samples? 

Line 435: Which is it, soak or co-crystallized? If both were tried, which led to the structure 
presented? 

Line 456-457 and rest of methods section: the preferred abbreviation for milliliter is mL not ml 
(same for microliter; uL not ul) 

Figure 2: It should be clearly stated what the control is. Just sialidase? 

Figure 3: Baccac_02680 structure should be labelled as the E143Q mutant. In panel b, why are the 
catalytic residues colored red in Bf4060 but not in Baccac_02680? Be consistent. Similar issue in 
sup fig 19. Check caption carefully – Beta1,3-linkage, “insert number here”, the supplementary 
figure you want to refer readers to is 19 not 17. 

Supplemental Information 
Typographical/grammatical/sentence-structure problems must be addressed on lines: 110, 166, 
226, 265, 301, 504 

Line 190-198: Please cut down on these texts which are redundant with lines 193-205 from the 
main text. The supplemental discussion should complement the main text instead of rephrasing 
the same thing. 

Line 209-213: These texts are likewise redundant with main text lines 181-192. 

Line 30-527: Carefully go through every section and remove all instances of redundancy with the 
main text. 

Line 316-323: I’m curious how convincing the electron density that supports skew boat 
conformation is at 2.7 A resolution for Baccac_02680. There is not a single figure with electron 
density maps; 2Fo-Fc maps at a minimum, and ideally composite omit maps, should be 
incorporated into figure. 

Line 316-323: Privateer [Nature Structural & Molecular Biology volume 22, pages 833–834 (2015)] 
must be used to validate these sugar models and the resulting output reported in a supplemental 
table. 

Line 321-323: So the density was too poor to define the ligand in BF4060 and it was built anyway? 
Same comments as previous: please show appropriate maps and run Privateer validation. 

Line 345-346: What does “different evolutionary origins” mean here? Presumably all enzymes in 
this subfamily have a common ancestor (hence the ability to construct a meaningful phylogeny)? 

Table 3: The “prefixes used for brevity” are really superfluous and should be replaced with the 
“true locus tag prefixes” throughout. In most cases they are the same, or do not result in the 
remove of many characters. Is this just laziness regarding figure editing? 

Table 5: Completely missing all statistics for apo Baccac_02680 and Baccac_02680 E143Q. For 
Amuc_0724 and BF4060 were there really zero water molecules? Missing Ramachandran stats for 
BF4060. Table caption: “reflections that were excluded from…..”. 

Table 6: What is L404 ligand? 

Figure 4: Please make the distinction between e) and f) clearer in the figure caption because they 
are both mixed-linkage glucan. Xyloglucan in i) is inaccurate. 

Figure 5: Label figure panels 

Figure 6 and 7: I am unable to find a look-up table as described in the legend. Regardless, most of 
the branch labels are impossible to read. Overall, these trees should be presented with an 
alternate layout that avoids overlap; a rectangular format would be far preferred, using collapsed 
branches where advantageous. A properly organised and displayed tree would not require all of 
the accessory text annotations and arrows. 

Figure 6 and 7: Insufficient information is provided regarding the algorithms used for sequence 
alignment and phylogeny estimation. Presently, there is no information on the statistical 
significance of any of the “Branches” described. At a minimum, a Maximum Likelihood phylogenies 
should be presented, using at least 100 bootstraps, and significant bootstrap values must be 
indicated on the published trees. A suitable outgroup should be used to root the trees; in the case 
of Figure 7, presumably one or more related GH16 subfamilies could be used. 

Figure 6 and 7: Please provide the input multiple sequence alignment files in FASTA format and 
trees in a suitable format (e.g. Newick, PhyloXML) as supplemental information files to allow 

reader direct access to the underlying data. 

Figure 8: The last sentence of the caption is slightly confusing. Please make it clear that BT0455 is 
the sialidase and GH95 is the fucosidase. 

Figure 11: Is species 14 correctly depicted? There is a stray GlcNAc on the end of a row of 
sulfations. 

Figure 13: Panel E is missing and the panel labels on the figure are not consistent with their 
description in the figure caption. Is low-activity Amuc_0875 the missing data here? 

Figure 15: The lanes are not labeled. 

Figure 16: μM enzyme not μm, it is a unit of concentration not length. 

Figure 16: Negative results determined only by TLC analysis is not particularly compelling, 
especially given weak staining efficiency (high limit of detection) and some streaking at Rf values 
consistent with oligosaccharides is observed. Supporting data using a standard reducing sugar 
activity assay must be provided to quantify relative specific activities. 

Figure 17: It would be helpful if the relevant loops are labeled with their finger number. Also, 
consider moving this figure to after 18 and 19 as its content is first referred to from the text after 
first reference to 18 and 19. 

End comments