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Recognition of Protein-Linked Glycans As a Determinant of Peptidase Activity

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Recognition of Protein-Linked Glycans As a Determinant of Peptidase Activity Recognition of protein-linked glycans as a determinant PNAS PLUS of peptidase activity Ilit Noacha, Elizabeth Ficko-Bleana,1, Benjamin Pluvinagea, Christopher Stuarta, Meredith L. Jenkinsa, Denis Brochub, Nakita Buenbrazoc, Warren Wakarchukc, John E. Burkea, Michel Gilbertb, and Alisdair B. Borastona,2 aDepartment of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6, Canada; bHuman Health Therapeutics, National Research Council Canada, Ottawa, ON K1A 0R6, Canada; and cDepartment of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada Edited by Chi-Huey Wong, Academia Sinica, Taipei, Taiwan, and approved December 20, 2016 (received for review September 9, 2016) The vast majority of proteins are posttranslationally altered, with the by mass, and the glycans tend to be very heterogeneous by virtue of addition of covalently linked sugars (glycosylation) being one of the differing extensions and branching of the core structures (6). The most abundant modifications. However, despite the hydrolysis of abundance and frequency of posttranslational modification to pro- protein peptide bonds by peptidases being a process essential to all teins, particularly large additions like glycans, suggests a need for life on Earth, the fundamental details of how peptidases accommo- peptidases to accommodate more than just the peptide component date posttranslational modifications, including glycosylation, has not of the substrate. Indeed, there is evidence that some peptidases are been addressed. Through biochemical analyses and X-ray crystallo- selective for heavily O-glycosylated proteins; however, the molecular graphic structures we show that to hydrolyze their substrates, three basis of how these enzymes recognize substrates is entirely unknown. structurally related metallopeptidases require the specific recognition Peptidases with known selectivity for heavily O-glycosylated of O-linked glycan modifications via carbohydrate-specific subsites proteins include O-sialylglycoprotease from Mannheimia haemo- immediately adjacent to their peptidase catalytic machinery. The lytica (formerly classified into the deleted MEROPS family M22) three peptidases showed selectivity for different glycans, revealing (7), StcE from Escherichia coli (family M66) (8), viral enhancins protein-specific adaptations to particular glycan modifications, yet (family M60) (9), and some serine protease autotransporters always cleaved the peptide bond immediately preceding the produced by enteric pathogens (SPATEs proteins; family S6) (10). glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular un- Similarly, BT4244 from Bacteroides thetaiotaomicron (11), IMPa BIOCHEMISTRY derstanding of glycoprotein degradation. from Pseudomonas aeruginosa (12), and SslE from E. coli (13) also display selectivity for glycosylated proteins. These three latter O-glycopeptidase | metallopeptidase | glycoprotein | O-glycosylation extracellular peptidases are members of the recently defined Pfam family PF13402 (also annotated as “peptidase M60, enhancin, and enhancin-like” or “M60-like”) (11). This family, which is in part eptidases catalyze the hydrolysis of the peptide bonds in Pproteins and, by this activity, play fundamental roles in an defined by the possession of a conserved HEXXH(8,24)E glu- enormous variety of critical biological processes across all of the zincin metallopeptidase motif (14), is distributed widely among taxonomic kingdoms. These enzymes can be classified by their prokaryotes and eukaryotes and was proposed by Nakjang et al. to catalytic mechanisms into seven groupings: serine (S), cysteine (C), threonine (T), aspartic (A), glutamic (G), asparagine (N), metallo Significance (M), or asparagine lyase (N) catalytic types (1). Each grouping of catalytic types has been broken into a robust classification of >240 Protein glycosylation is one of the most abundant and important families, which are based on amino acid sequence identity, posttranslational modifications where the protein-linked glycans whereas the categorization of >50 clans reflects the structural can impart specific physiochemical properties to the glycoprotein similarities between the families (https://merops.sanger.ac.uk)(1). and/or the glycans themselves can mediate particular biological This classification contains >0.9 million putative peptidase se- functions. The degradation of glycosylated proteins in normal or quences but only <0.5% of these have been experimentally veri- pathogenic processes, therefore, is an important biological pro- fied, suggesting that we are far from a complete understanding of cess. This study reveals the molecular basis of how peptidases how this vast sequence space translates into variable substrate can use the O-glycans present on glycoproteins as a critical de- specificities and biological outcomes. terminant of peptidase activity and, in doing so, provides unique With billions of sequences for known or putative proteins in the insight into how peptidases may directly use posttranslational protein databases, the variety of possible peptidase substrates is vast. modifications present on their substrates to influence recogni- For example, the human genome encodes for ∼600 peptidases, yet tion and peptide bond cleavage. over 17,000 genes are known to be transcribed and translated into unique protein products that are possible substrates for the encoded Author contributions: I.N., E.F.-B., and A.B.B. designed research; I.N., E.F.-B., B.P., C.S., M.L.J., D.B., N.B., W.W., J.E.B., M.G., and A.B.B. performed research; D.B., N.B., W.W., and M.G. peptidases (2, 3). This highlights one of the major challenges in contributed new reagents/analytic tools; I.N., E.F.-B., B.P., M.L.J., J.E.B., and A.B.B. ana- characterizing peptidase specificity and identifying specific substrates lyzed data; and I.N. and A.B.B. wrote the paper. (4). Furthermore, the diversity in substrates is substantially increased The authors declare no conflict of interest. by posttranslational modifications. Indeed, upwards of 50% of pro- This article is a PNAS Direct Submission. teins are thought to be posttranslationally modified with glycosyla- Data deposition: The atomic coordinates and structure factors have been deposited in the tion being one of the most abundant alterations (5). Typically, Protein Data Bank, www.pdb.org (PDB ID codes 5KD2 for BT4244_E575Q_l; 5KD5 for protein glycosylation constitutes glycans attached to asparagine side BT4244_m; 5KD8 for BT4244-TnAg; 5KDJ for ZmpB_l; 5KDN for ZmpB_m; 5KDS for chains (N linked) or serine/threonine side chains (O linked). The ZmpB-BSMfrg; 5KDU for ZmpB-STAg; 5KDV for IMPa-6His; 5KDW for IMPa; and 5KDX for IMPa-TAg). structures of glycans attached to a given site can be variable, giving 1Present address: Sorbonne Universités, UPMC Université Paris 06, UMR 8227, Integrative glycoforms of a protein. For example, there are eight distinct core Biology of Marine Models, Station Biologique de Roscoff, CS 90074, 29688 Roscoff O-glycan structures, all covalently α-linked via an N-acetylgalactos- CEDEX, France. amine (GalNAc) moiety to the hydroxyl group of serine or threonine 2To whom correspondence should be addressed. Email: [email protected]. side chains (6). Furthermore, the degree of glycosylation is protein This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dependent, but the average mucin protein is ∼50% O-linked glycans 1073/pnas.1615141114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1615141114 PNAS | Published online January 17, 2017 | E679–E688 Downloaded by guest on September 30, 2021 comprise glycoprotein-specific peptidases (11). Although the Pfam Results classification of these three peptidases indicates an evolutionary re- In Vitro Peptidase Activity. Our target proteins possessed complex lationship between them, BT4244, IMPa, and SslE are classified into multimodularity (SI Appendix,Fig.S1); thus, to advance their the separate MEROPS families M60, M88, and M98, respectively. study, gene fragments encoding the M60-like domains of BT4244 Through their ability to cleave O-glycoproteins, many of these and ZmpB (referred to as BT4244_m and ZmpB_m, respectively) peptidases have important roles in cell biology (10, 12, 15), have were expressed in E. coli. IMPa was similarly produced but as a been implicated in bacterial pathogenesis (16–18), or have found full-length protein. Treatment of highly O-glycosylated bovine use as reagents (7). Despite the functional significance of O-gly- submaxillary mucin (BSM) with BT4244_m resulted in a clear coprotein degrading peptidases, however, their mode of action change in the electrophoretic mobility of BSM, whereas IMPa and on O-glycoproteins is unidentified. To obtain insight into how ZmpB_m gave nearly complete clearing of the BSM (Fig. 1A). We peptidases select for glycosylated substrates, we focused our also used a microtiter plate-based mucinase assay using immobi- studies on the model examples BT4244, IMPa, and ZmpB (locus lized biotinylated BSM as a substrate and all three peptidases also tag CPF_1489) from Clostridium perfringens (strain ATCC 13124), displayed activity in this assay (SI Appendix,Fig.S2). These results were consistent with the reported activity of BT4244 and IMPa on the latter of which is a member of the Pfam M60-like family but is glycoprotein substrates (11, 12), while revealing comparable not classified into a MEROPS family. Biochemical
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