Peer review of: Trefoil factors share a lectin activity that defines their role in mucus (2019)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

Major points 

 

Major concern refers to novelty and priority of claims. Lectin function and specificity of TFF2 were 
previously reported (J. Biol. Chem. 2014,289: 27363-27375), and implications from this study refer to 
structural details on both sides, the ligand and its receptor. Due to the structural similarities of TFFs, 
in particular with respect to the primary sequences in loop 3 (with the highly conserved Trp45 and 
Phe36 residues) and loop 2, which together form the lectin binding pocket, similar binding 
characteristics could be expected for TFF1 and TFF3. The latter TFFs are in the focus of the current 
paper. The work presented is solid and the conclusions drawn are supported by the provided 
evidence. A further argument against the novelty issue is however provided by authors themselves, 
as they pre-published the not peer-reviewed paper and uploaded it on Research Gate. 

In this context, authors do not adequately refer to the previous work. Reference is made in the 
appending literature list, but the text refers only to “These observations lie in contrast to a report 
that TFF2 interacts with an α-DGlcNAc-terminated O-glycan from mucin“. The impression is that 
authors try to hide in a way the merits of the previous publication by concealing that the lectin 
specificity of TFF2 has been characterized in detail and on a solid physico-chemical level (covering 
solid-phase binding to neoglycolipids, mass spectrometry and NMR. No further reference is made in 
the discussion to this paper suggesting to reviewers and readers that the current paper claims 
priority. On the other hand, authors start the presentation of results with the sentence “To establish 
that TFF1 and TFF3 possessed the same α-GlcNAc-dependent binding activity as TFF2,...”, which 
means that their work actually extends previous work. What authors can claim actually is the 
extension of the existing evidence by providing proof of lectin function for other TFFs (TFF1 and 
TFF3) with similar specificity. These facts need to be clearly stated at prominent position (Abstract, 
Introduction, Discussion). 

 

p2/3, lines 47-53: Moreover, the statement that the lectin function of TFF2 contrasts with a large 
number of binding partners is weak, as it ignores possible other mechanisms of interaction. Besides 
a lectin- carbohydrate interaction, TFFs, like TFF2, could bind to other proteins via hydrophobic 
interaction. Authors should refer to the paper by Bonar, J. Biol. Chem. 2014, 289: 29677-29690, who 
provided evidence for the existence of a hydrophobic patch within and at the border of the lectin 
binding pocket of TFF2. 

Also other important references are missing, which point to alternative aspects in host pathogen 
interactions. 

 

Minor points 

There is a need to define terms like “type III mucin-like O-glycans”. This term is not generally used in 
the field and could refer to blood group structures or to core types or something else. 

The claimed minimum structure for binding of TFF lectins was previously reported in JBC 2014 (refer 
to the solid phase binding assays on neoglycolipids derived from partial structures of the branched 
mucin glycans. However, it was found to be much less active compared to the trisaccharide 
GlcNAca1-4Galb1-4GlcNAc in binding assays on neoglycolipids. 

Lectin-mediated binding of TFFs to MUC5AC is questionable, as this mucin has not been shown to 
express alpha-GlcNAc terminating glycans. The latter is a feature of deep gastric glands in the 
stomach expressing MUC6. 

 

Franz-Georg Hanisch 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

This study by Jarva et al demonstrates that recombinant TFF1, TFF2 and TFF3 all bind the GlCNAc-α-
1,4-Gal disaccharide. Using an ELISA binding of monomeric TFF1 and TFF3 to a commercial source of 
porcine gastric mucin and to the same mucin which had undergone reduction and alkylation but still 
possesses the α -GlcNac terminated structure was demonstrated. Treatment of the mucin with an α-
D-N-acetylglucosaminidase from Clostridium perfringens abrogated binding, thus demonstrating 
that TFF1 and TFF3 both bind to an α-GlcNac structure present on mucin similar to what has been 
reported previously for TFF2 by Hanisch et al. (Ref#20). Isothermal calorimetry and tryptophan 
fluorescence quenching determined that no binding could be detected to simple monosaccharides 
but monomeric TFF1 and 3 and TFF2 bound to GlcNAC- α-1,4-Gal disaccharide, a terminating 
structure found on Type III mucins. Interestingly no binding was detected with other α-1,4 linked 
disaccharides such as maltose. Crystallisation of the monomeric TFF-GlcNAc-α-1,4-Gal complex 
indicated that the disaccharide binds within a hydrophobic cleft. Asp20 and Trp47 residues on 
mTFF3 make direct contact with the glycan. However, in TFF1 and TFF2 an ASn is found in place of 
Asp 20. Mutagenesis of these Asp/ASn and Trp resiudes in dimeric forms of TFF1 and TFF3 reduced 
the affinity of TFF1 for mucin and inhibited completely binding of TFF3. Tryptophan fluorescence 
quenching assays at different pHs suggest that the conserved Asn20 is important for binding of TFF1 
and TFF2 at low pH. The dimeric forms of TFF1 and TFF3 were required for mucin agglutination. 
Mutant dimers unable to bind GlcNAc- α-1,4-Gal disaccharide were unable to agglutinate mucin. 
Treatment of mucin containing biotinylated versions of TFF1 and TFF3 with α-D-N-
acetylglucosaminidase liberated TFF1 and TFF3 from the mucin. Finally, a phylogenetic analysis of 
proteins known to have trefoil domains suggests that they may also serve as lectins. 

 

This is a very interesting study on a group of proteins with obvious therapeutic potential, whose 
mode of action has remained elusive for a long time. Identification of a binding partner for all three 
trefoil peptides and evidence of how that mediates binding to mucins would be of interest to a large 
number of investigators and should stimulate further research into these proteins. 

 

Previous studies have shown that the trefoil peptides have lectin activity. Structural studies 
identified a hydrophobic cleft on TFFs capable of accommodating either an oligosaccharide or an 
aromatic amino acid side chain (Polshakov et al., 1997, J. Mol. Biol. 267: 418–432). 
Lipopolysaccharide of the gastric pathogen <i>Helicobacter pylori</i> was identified as a binding 
partner for TFF1 (Reeves et al., 2008, Ref #23). More recently Hanisch et al., 2014 (ref# 20), 
demonstrated that TFF2 bound α-GlcNac and Dunne et al, (Microorganisms. 2018 May 18;6(2). pii: 
E44. doi: 10.3390/microorganisms6020044) reported a striking correlation between binding of TFF1 
to human gastric mucins and binding of <i>Griffonia simplicifolia</i> lectin-II which is specific for 
terminal non reducing α- or β- linked N-acetylglucosamine. While some of these references are 
mentioned in the discussion it would be informative if a short summary of previous work showing 
that TFFs have lectin activity was clearly stated in the introduction. 

 

The evidence that the recombinant proteins produced for this study bind the GlcNAC-α-1,4-Gal 
disaccharide is convincing. However, reactivity of the recombinant proteins produced with 
antibodies raised against different epitopes of the trefoil proteins would be convincing evidence that 
these proteins are representative of native proteins. If this were possible it would compliment the 
SDS PAGE results nicely. 

 

While Figure 2a shows that dTFF1<sub>bio</sub>N14A has reduced affinity for mucin considerable 
binding still occurs in contrast to binding of dTFF3<sup>bio</sub>-D20A. This suggests that TFF1 
may bind to other glycans present on the mucin. Use of a glycan array to compare binding of TFF1, 
TFF2 and TFF3 to different glycans could be done to assess if the GlcNAC- α-1,4-Gal disaccharide is 
the only glycan present on mucins that the TFFs bind to. How specific is the <i>Cp</i>GH899enzyme 
used to treat the mucin? Is it possible it could cleave other glycans. Considering the site-specific 
location of TFFs and the association of different TFFs with different mucins it would not be surprising 
to find that they each may have some unique binding partners. 

 

Have the authors considered the multivalent presentation of glycans on mucins and how important 
this might be for TFF binding to mucins. Perhaps this could be discussed in the paper. 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

In this work, Jarva and colleagues demonstrate that demonstrate that all trefoil factors (TFFs) are 
divalent lectins that recognize the GlcNAc-α-1,4-Gal disaccharide, which terminates type-III mucin-
like O-glycans. The authors report crystal structures of the unliganded form of HsTFF1 and that of 
HsTFF3 in complex with GlcNAc-a-1,4-Gal, at 2.40 and 1.55 A resolution, respectively. In combination 
with mutagenic and biophysical data provided they provide insights into how the TFF peptides 
recognize the GlcNAc-α-1,4-Gal disaccharide and rationalize their ability to modulate the physical 
properties of mucus across different pH ranges. In principle, the manuscript is therefore suitable for 
publication in Nature Communications in terms of its novelty and importance. However, the 
manuscript can be improved, with several aspects of the work requiring clarifications: 

 

1. page 3, line 60: “To establish that TFF1 and TFF3 possessed the same α-GlcNAc-dependent binding 
activity as TFF2…” I would suggest to perform the TFF2 binding activities as a control and introduce 
the results in Figure 1c. 

 

2. page 3, line 63: “reduced and alkylated pMucin (pMucinred), which is denatured…”. Please 
provide biophysical data or reference in support of this statement. 

 

3. page 4, line 84: Please, prepare two Figures: Figure 1, including panels (a) to (e). Please, increase 
the size of panels (a), (c), and (d). Figure 2, including panels (f) to (k). Please show the overall 
structure of TFF1, TFF2 and TFF3. Stereo views. Please, introduce an electrostatic surface 
representation for the TFF3–GlcNAc-a-1,4-Gal complex. Please, remove panel (h). 

 

4. Table S2. Please, include the following information: (i) reflections used in refinement, (ii) 
reflections used for R-free, (iii) CC(work), (iv) CC(free), (v) clashcore. 

 

5. page 4, line 93: Please, provide overall superposition of the CBM32 domain (PDB ID: 4A6O) in 
complex with GlcNAc-a-1,4-Gal with HsTFF3. 

 

6. Can the authors speculate on the molecular mechanism by which TFFs increase the viscoelasticity 
of the mucosa? 

 

I support publication of the manuscript in the case all these questions can be addressed 
satisfactorily.