Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains

Langsford, M.L.; Gilkes, Neil R.; Singh, Bhagirath; Moser, Bernhard; Miller, Robert C.; Warren, R. A. J.; Kilburn, Douglas G.

doi:10.1016/0014-5793(87)81150-x

Cited by 150 publications

(101 citation statements)

References 13 publications

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“…The secretion of proteases aids in the competition for resources and also is a means for pathogen defense mechanisms (46). It is clear from previous studies that glycans can protect against proteolysis (20,42). Our results also show such protection, showing that glycans can protect the peptide backbone from proteolytic attack, likely through a steric hindrance mechanism.…”

Section: Discussionsupporting

confidence: 67%

“…Glycosylation can modulate both the physical and biological properties of proteins (39,40) and aid in protein folding and secretion (41). Indeed, O-linked glycans on cellulase linkers confer proteolytic resistance (20,42) and have been shown to impart affinity to crystalline cellulose (21). Furthermore, it has been shown that small O-linked glycans exist on the TrCel7A CBM near the binding face ( Fig.…”

Section: Discussionmentioning

confidence: 99%

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Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Chen

Drake

Resch

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

154

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The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16°C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. chemical synthesis | cellulase | biofuels | protein engineering

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Chen

Drake

Resch

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

154

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“…In particular, the enzymatic activity of isolated CexCD toward cleavage of soluble substrates is similar to that of the full-length Cex, with or without linker glycosylation (32,54). Thus, neither the linker nor the cellulose-binding module alters the catalytic properties of the catalytic domain of Cex.…”

Section: Cex Is Composed Of Independent Catalytic and Cellulosebindinmentioning

confidence: 99%

“…Glycosylated Cex from S. lividans is reported to contain ϳ17 mol of mannose, with traces of galactose, per mole of protein (30,31). These differences are minor because samples of O-glycosylated Cex from C. fimi and S. lividans have very similar kinetic properties and are both resistant toward proteolytic degradation (31,32). Based on chemical shift, 15 N relaxation, and amide residual dipolar coupling (RDC) measurements, we demonstrate that the catalytic and cellulose-binding domains are physically independent and joined by a PT linker that is conformationally dynamic on the ns-to-ps time scale.…”

mentioning

confidence: 99%

Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis

Poon

Withers

McIntosh

2007

Journal of Biological Chemistry

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The modular xylanase Cex (or CfXyn10A) from Cellulomonas fimi consists of an N-terminal catalytic domain and a C-terminal cellulose-binding domain, joined by a glycosylated proline-threonine (PT) linker. To characterize the conformation and dynamics of the Cex linker and the consequences of its modification, we have used NMR spectroscopy to study full-length Cex in its nonglycosylated (ϳ47 kDa) and glycosylated (ϳ51 kDa) forms. The PT linker lacks any predominant structure in either form as indicated by random coil amide chemical shifts. Furthermore, heteronuclear 1 H-15 N nuclear Overhauser effect relaxation measurements demonstrate that the linker is flexible on the ns-to-ps time scale and that glycosylation partially dampens this flexibility. The catalytic and cellulose-binding domains also exhibit identical amide chemical shifts whether in isolation or in the context of either unmodified or glycosylated full-length Cex. Therefore, there are no noncovalent interactions between the two domains of Cex or between either domain and the linker. This conclusion is supported by the distinct 15 N relaxation properties of the two domains, as well as their differential alignment within a magnetic field by Pf1 phage particles. These data demonstrate that the PT linker is a flexible tether, joining the structurally independent catalytic and cellulosebinding domains of Cex in an ensemble of conformations; however, more extended forms may predominate because of restrictions imparted by the alternating proline residues. This supports the postulate that the binding-domain anchors Cex to the surface of cellulose, whereas the linker provides flexibility for the catalytic domain to hydrolyze nearby hemicellulose (xylan) chains.Cellulolytic organisms produce a battery of endo-and exoglucanases necessary for the hydrolysis of cellulose and hemicellulose. These glycoside hydrolases are typically modular, consisting of conserved catalytic and carbohydrate-binding domains (or modules), as well as possible ancillary domains, joined by variable linker sequences (1-3). In general, the constituent domains of glycoside hydrolases are structurally independent and exhibit some aspects of their respective functions when separated (4 -6). Thus, binding modules appear to facilitate catalysis by targeting and maintaining the proximity of the catalytic domain toward substrates within complex macromolecular systems, such as the plant cell wall, as well as through possible disruptive effects on the structures of the polysaccharides within these systems (7,8).The synergistic activity of the catalytic and carbohydratebinding domains in a glycoside hydrolase requires that they be covalently tethered to one another via a linker sequence of the appropriate length and/or flexibility. The functional importance of these interdomain linkers, which can range from only a few to over a hundred residues and are often rich in proline and hydroxyamino acids (1), has been established largely through deletion studies (9 -12). However, the physical properties of g...

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“…Generally, it has been reported that glycosylation protects proteins from proteolysis. 28) In plant pathogens, protection of secreted enzymes from proteolysis might play an especially important role in the success of pathogen development in planta. 29) Another possible role of the N-terminal sequences of Bx-ENG-2 and Bx-ENG-3 is to contribute to forming a subunit, since Bx-ENG-2 and Bx-ENG-3 existed in a dimeric form, whereas Bx-ENG-1 without the extra sequence existed in a monomeric form.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of Recombinant Endoglucanases from the Pine Wood NematodeBursaphelenchus xylophilus

Shibuya¹,

Kikuchi²

2008

Bioscience, Biotechnology, and Biochemistry

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Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains

Cited by 150 publications

References 13 publications

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis

Purification and Characterization of Recombinant Endoglucanases from the Pine Wood NematodeBursaphelenchus xylophilus

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