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Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases

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Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases International Journal of Molecular Sciences Review Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases Peter Goettig Structural Biology Group, Faculty of Molecular Biology, University of Salzburg, Billrothstrasse 11, 5020 Salzburg, Austria; [email protected]; Tel.: +43-662-8044-7283; Fax: +43-662-8044-7209 Academic Editor: Cheorl-Ho Kim Received: 30 July 2016; Accepted: 10 November 2016; Published: 25 November 2016 Abstract: Posttranslational modifications are an important feature of most proteases in higher organisms, such as the conversion of inactive zymogens into active proteases. To date, little information is available on the role of glycosylation and functional implications for secreted proteases. Besides a stabilizing effect and protection against proteolysis, several proteases show a significant influence of glycosylation on the catalytic activity. Glycans can alter the substrate recognition, the specificity and binding affinity, as well as the turnover rates. However, there is currently no known general pattern, since glycosylation can have both stimulating and inhibiting effects on activity. Thus, a comparative analysis of individual cases with sufficient enzyme kinetic and structural data is a first approach to describe mechanistic principles that govern the effects of glycosylation on the function of proteases. The understanding of glycan functions becomes highly significant in proteomic and glycomic studies, which demonstrated that cancer-associated proteases, such as kallikrein-related peptidase 3, exhibit strongly altered glycosylation patterns in pathological cases. Such findings can contribute to a variety of future biomedical applications. Keywords: secreted protease; sequon; N-glycosylation; O-glycosylation; core glycan; enzyme kinetics; substrate recognition; flexible loops; Michaelis constant; turnover number 1. Introduction Glycosylation is a posttranslational modification that is found on about 50% of all proteins, in particular on secreted and transmembrane proteins of eukaryotes, archaea and to a lesser extent in prokaryotes [1–3]. Eukaryotic proteins require glycosylation for proper folding, oligomerization and solubility, while glycans significantly prolong the stability and half-life time in many cases by protection against proteolysis [4,5]. Although N-glycosylation is more frequent, O-glycosylation can similarly protect against general and specific proteolysis [6–8]. Protein trafficking, i.e., the sending of proteins to cellular compartments or to the extracellular matrix, depends on specific, covalently linked glycans [9]. In addition, glycans play an important role in the interaction and recognition of proteins, such as in the context of immunity and cell adhesion [10–12]. Glycosylation may even protect against molecular damage by free radicals [13]. In recent years, increasing evidence was found that glycans have distinct effects on the activity of many enzymes, in particular as regulatory modules for substrate binding and turnover. This study gives an overview on the most relevant types of glycosylation of proteases, regarding the structural knowledge and the functions of glycans. The importance of this little investigated field lies in the enormous diversity of possible glycosylation variants and the altered functionality of proteins under healthy or disease conditions. N-glycosylation at sequons of the Asn-Xaa-Ser/Thr type is widespread in proteins of archaea and eukaryotes, whereby proline is largely excluded as Xaa and disfavored as residue following Ser/Thr [14,15]. Some rare sequons are Asn-Xaa-Cys (1%), Asn-Gly (0.5%) and Asn-Xaa-Val (<0.5%) [16]. The process of N-glycosylation is extensively described in the literature on glycobiology [17]. Essentially, a newly Int. J. Mol. Sci. 2016, 17, 1969; doi:10.3390/ijms17121969 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 1969 2 of 24 Int. J. Mol. Sci. 2016, 17, 1969 2 of 23 synthesized polypeptide emerging from a ribosome binds with a signal peptide to a signal recognition Essentially, a newly synthesized polypeptide emerging from a ribosome binds with a signal peptide particle,to a signal which recognition docks to a receptorparticle, inwhich the endoplasmic docks to a reticulumreceptor in (ER) the membrane endoplasmic and formsreticulum a complex (ER) withmembrane the Sec and machinery, forms a complex which transferswith the Sec the machin polypeptideery, which through transfers a transmembrane the polypeptide channel through into a thetransmembrane lumen of the channel ER [18 into,19]. the A lumen signal of peptidase the ER [1 cleaves8,19]. A thesignal N-terminal peptidase signalcleaves peptide the N-terminal and the oligosaccharyltransferase complex attaches a GlcNAc Man Glc precursor at a suitable sequon of signal peptide and the oligosaccharyltransferase complex2 attaches9 3 a GlcNAc2Man9Glc3 precursor at thea suitable Asn-Xaa-Ser/Thr sequon of typethe Asn-Xaa-Ser/Thr [20]. Subsequently, type the [20].N-glycosylated Subsequently, polypeptide the N-glycosylated folds in thepolypeptide oxidizing environmentfolds in the oxidizing of the ER, environment supported of by the protein ER, suppo disulfiderted by isomerase protein disulfide for disulfide isomerase formation for disulfide and by variousformation chaperones and by various [21,22]. chaperones Afterwards, [21,22]. glucosidases Afterwards, and mannosidases glucosidases and trim mannosidases the N-glycan precursortrim the toN Man-glycan5GlcNAc precursor2 or GlcNAcManto Man5GlcNAc3GlcNAc2 or GlcNAcMan2 core glycans,3GlcNAc which2 core are extendedglycans, which by glucosyltransferases, are extended by accompaniedglucosyltransferases, by protein accompanied quality control by protein and followed quality by control sorting and and followed further processingby sorting onand their further way throughprocessing the on ER their Golgi way intermediate through the compartmentER Golgi intermediate into the compartment Golgi [23,24]. into Final the modifications Golgi [23,24]. Final of the Nmodifications-glycans in the of Golgi the N comprise-glycans in extensions the Golgi by comprise transferases extens thations attach by transferasesN-acetyl-glucosamine that attach N (GlcNAc),-acetyl- fucose,glucosamine galactose, (GlcNAc), mannose, fucose, and sialicgalactose, acid sugars,mannose, before and sortingsialic acid to secretorysugars, before vesicles sorting [25]. to Variations secretory of branchingvesicles [25]. generate Variations a large diversityof branching of N -glycansgenerate with a large distinct diversity composition of N under-glycans physiological with distinct and pathologicalcomposition conditions under physiological (Figure1A) and [26 pathological]. conditions (Figure 1A) [26]. FigureFigure 1.1. ExamplesExamples of the most relevant types types of of glyc glycosylationosylation according according to to the the literature literature [3,27]. [3,27 ]. (A(A)) NN-glycosylation-glycosylation ofof asparagineasparagine inin sequonssequons withwith thethe consensusconsensus sequence Asn-Xaa-Ser/Thr. Asn-Xaa-Ser/Thr. N-glycans are generated by trimming and extending the common precursor GlcNAc2Man9Glu3. Small N-glycans are generated by trimming and extending the common precursor GlcNAc2Man9Glu3. Smallcore glycans core glycans are mostly are mostly intermediates intermediates in mammalia in mammaliann glycan glycan synthesis, synthesis, bu butt often often occur occur in in more more primitiveprimitive eukaryotes eukaryotes andand insects,insects, asas usedused for recombinant expression. Mammalian Mammalian NN-glycans-glycans exhibit exhibit anan enormous enormous diversity,diversity, duedue toto manymany possiblepossible combinationscombinations of branching sugars; sugars; ( (BB)) OO-glycosylation-glycosylation atat SerSer and Thr Thr is is found found in inall allkingdoms kingdoms of life. of life.There Thereis no distinct is nodistinct consensus consensus sequence, sequence, but proline- but proline-richrich regionsregions are favo arered, favored, e.g., a typical e.g., a typicalO-glycanO -glycansite would site be would Pro-Ser/Thr-Xaa-Yaa-Pro. be Pro-Ser/Thr-Xaa-Yaa-Pro. A very Acommon very common mammalian mammalian O-glycanO-glycan is the ismucin-type the mucin-type that starts that startswith withGalNAc GalNAc and is and extended is extended by bygalactose galactose and and sialic sialic acids acids or or GlcNAc, GlcNAc, with with eight eight different different cores cores known. known. In In addition, addition, the the OO-xylose-xylose linked,linked, non-branchednon-branched glucosamineglucosamine glycans (GAG) (GAG) or or proteoglycans proteoglycans are are a a large large and and diverse diverse glycan glycan family.family. The The displayed displayed chondroitinchondroitin cancan bebe phosphorylated and heavily heavily su sulfated,lfated, comprising comprising up up to to fifty fifty disaccharidedisaccharide units. units. OO-GlcNAc-GlcNAc glycansglycans occuroccur inside cells, even even in in the the nucleus, nucleus, while while OO-galactosylation-galactosylation isis found found at at hydroxylysine hydroxylysine residuesresidues (Hyl)(Hyl) ofof collagens.collagens. Int. J. Mol. Sci. 2016, 17, 1969 3 of 24 Earlier structural database analyses reported a relatively low percentage of only 27% N-glycosylation of all sequons in human proteins, of which 96% belonged to secreted and membrane proteins and 4% to cytoplasmic and nuclear proteins [28,29]. More recent data suggested around 85% occupancy of sequons, with 50% glycosylation of all proteins
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