Analysis of a New Family of Widely Distributed Metal-independent α-Mannosidases Provides Unique Insight into the Processing of N-Linked Glycans

Abstract

The modification of N-glycans by ␣-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for ␣1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-␣1,6-mannosidase activity consistent with specificity for N-linked glycans having their ␣1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substratebound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (␣/␣) 6 -fold, recognize and hydrolyze the ␣1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-␣1,6-mannosidases.A feature of emerging importance to bacteria that colonize or infect humans is their capacity to process host glycans. Streptococcus pneumoniae is one notable human pathogen that relies on this ability for its full virulence (1). Among its known carbohydrate active virulence factors are NanA, StrH, BgaA, and EndoD. NanA Glycoside hydrolases, enzymes that break glycosidic bonds through a hydrolytic mechanism, are presently classified into 123-amino acid sequence based families (2). ␣-Mannosidases known to process N-glycans are found in families 38, 47, 76, 92, and 99. Very recent studies have shown the bacterial family 38 ␣-mannosidase from Streptococcus pyogenes (SpyGH38) to be a specific exo-␣1,3-mannosidase that is tolerant of the ␣1,6-branches in N-glycans (3). Analysis of family 92 glycoside hydrolases from the human gut symbiont Bacteroides thetaiotaomicron revealed an expanded repertoire of ␣-mannosidases (4). These enzymes displayed activity primarily toward ␣1,2-and ␣1,3-mannosidic linkages with some having low ␣1,6-mannosidase activity. In addition to the established ability of S. pneumoniae to exo-hydrolytically process the distal arms of complex glycans, which comprise sialic acid, galactose, and N-acetylglucosamine, consideration of additional putative carbohydrate-active enzymes found in this organism suggests it can partly degrade the mannose component of N-glycans using enzymes similar to those found in S. pyogenes and B. thetaiotaomicron. Through these observations it has become clear that some bacteria, possibly including S. pneumoniae, have the capacity to process the mannose component of N-glycans. A noteworthy gap, however, in the known bacterial N-glycan de...

The N-Glycan Cluster from Xanthomonas campestris pv. campestris

“…The plasmid expressing nixF (predicted to code for an endo-␤-N-acetylglucosaminidase, GH18) did not complement the activity of the ⌬nixE-nixL strain on pNP-␤-GlcNAc, consistent with the endo-activity predicted for this enzyme (data not shown). Similarly and as reported for enzymes belonging to the GH125 family (19), the plasmid expressing nixJ (predicted to code for an ␣-1,6-mannosidase, GH125) was not active on pNP-␣-Man (data not shown). Activity on pNP-Fuc was 11-fold lower when NixE was expressed from pC-nixE as compared with expression from pC-nixE-nixL ( Fig.…”

“…More recently, enzymes from Streptococcus pyogenes and Streptococcus pneumoniae classified in the GH38 (␣-1,3-mannosidase) and GH125 (␣-1,6-mannosidase) families, respectively, have been identified. These enzymes are active on N-glycans, highlighting the processing of N-glycans by Streptococcus bacteria (18,19). Finally, it was shown that the human pathogen Capnocytophaga canimorsus deglycosylates surface glycoproteins from the host and supports its growth on the released glycan moiety (20).…”

Structure and activity of theStreptococcus pyogenesfamily GH1 6-phospho-β-glucosidase SPy1599

“…SPy1604 is an -mannosidase belonging to GH38; it shows specificity for -1,3-and -1,6-mannosyl linkages (Suits et al, 2010), which are frequently associated with N-glycans. SPy1603 shows 62% identity (BLAST; http:// blast.ncbi.nlm.nih.gov) to a GH125 -mannosidase from S. pneumoniae (SpGH125; Gregg et al, 2011). The SpGH125 protein shows exo--1,6-mannosidase activity, which is again consistent with specificity for N-linked glycans.…”

Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase

“…In retaining GH38 (32), and inverting GH47 (33) GH92 (31) α-mannosidases, a divalent metal ion (Zn 2þ for GH38, Ca 2þ for GH47 and GH92) is found to coordinate the 2-and 3-hydroxyls, possibly assisting in catalysis through substrate distortion and in delivery of the nucleophile (31). By contrast, in the inverting GH125 enzymes no metal ion is observed in any structures and no substrate distortion was seen in a pseudo-Michaelis complex with an S-linked disaccharide spanning the active site (34). It is unclear how this family overcomes the destabilizing diaxial interaction of the attacking water nucleophile, although it is possible that a favorable hydrogen bond between the 2-OH of the sugar and the nucleophilic water may assist in nucleophilic attack in a similar way to the divalent metals in the GH38, GH47, and GH92 enzymes.…”