Identification and glucan-binding properties of a new carbohydrate-binding module family

2001 ◽  
Vol 361 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
Mazyar GHAFFARI ◽  
R. Antony J. WARREN ◽  
Douglas G. KILBURN
Keyword(s):  
Association Constant ◽  
Association Constants ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Bacillus Sp ◽  
Binding Properties ◽  
Alkalophilic Bacillus ◽  
Binding Subsites

The C-terminal 191-residue module of Cel5A from the alkalophilic Bacillus sp. 1139 comprises a carbohydrate-binding module (CBM) belonging to a previously unidentified family that we have classified as CBM family 28. This example, called CBM28, bound specifically to cello-oligosaccharides and mixed β-(1,3)(1,4)-glucans (barley β-glucan) with association constants of approximately (1–4)×104 M−1. Its binding to barley β-glucan was remarkably insensitive to pH between 7.0 and 10.9, in keeping with its alkalophilic source. CBM28 bound to cellulose having a significant non-crystalline content with an association constant similar to that for its binding to soluble glucans. CBM17 (CBM family 17) and CBM28 modules naturally occur as tandems. The CBM17/CBM28 tandem from Cel5A bound with apparent co-operativity to barley β-glucan. The association of CBM28 with cello-oligosaccharides was driven enthalpically and marked by the different thermodynamic contribution of three putative binding subsites that accommodate a cellohexaose molecule.

Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis

2001 ◽  
Vol 358 (2) ◽  
pp. 423-430 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
R. Antony J. WARREN ◽  
Douglas G. KILBURN
Keyword(s):  
Pichia Pastoris ◽  
Association Constant ◽  
Mutational Analysis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Glycosylation Sites ◽  
High Mannose

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc2Man8 to GlcNAc2Man14. The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (Ka) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The Ka of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.

scholarly journals Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

2007 ◽  
Vol 406 (2) ◽  
pp. 209-214 ◽  
Author(s):  
Lavinia Cicortas Gunnarsson ◽  
Cedric Montanier ◽  
Richard B. Tunnicliffe ◽  
Mike P. Williamson ◽  
Harry J. Gilbert ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Hydrophobic Interactions ◽  
Molecular Engineering ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Rhodothermus Marinus ◽  
Binding Properties ◽  
Carbohydrate Binding Modules

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.

scholarly journals Immunochemical specificity of the combining site of murine myeloma protein CAL20 TEPC1035 reactive with dextrans.

1981 ◽  
Vol 153 (1) ◽  
pp. 166-181 ◽  
Author(s):  
S Sugii ◽  
E A Kabat ◽  
M Shapiro ◽  
M Potter
Keyword(s):  
Binding Constant ◽  
Association Constant ◽  
Association Constants ◽  
The Other ◽  
Side Chains ◽  
Binding Properties ◽  
Myeloma Protein ◽  
Affinity Electrophoresis ◽  
Inhibition Data ◽  
Type Site

The immunochemical specificity of the combining sites of murine myeloma protein CAL20 TEPC1035 was studied by quantitative precipitin and precipitin inhibition assays. Myeloma protein CAL20 TEPC1035 precipitated with only three dextrans, B1355S4, B1498S, and B1501S, with high proportions of alpha(1 leads to 3) linkages, but not with any other dextrans, glycogen, and pullulan. Inhibition tests with various sugars show that the combining site of myeloma protein CAL20 TEPC1035 is most complementary to panose, a trisaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc. Panose was 3.3 times more potent than a tetrasaccharide DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 4)DGlc alpha(1 leads to 4)DGlc and 8, 23, 42, > 42 times more active than maltose, nigerose, isomaltose, and kojibiose, respectively. These findings were paralleled by their binding properties as determined by affinity electrophoresis. The association constants (Ka) of these three dextrans to myeloma protein CAL20 TEPC1035 ranged from 3.8 X 10(3) ml/g to 5.02 X 10(3) ml/g. The association constant of inhibitor (Kia) of panose was 8.19 X 10(3) M-1. Myeloma protein CAL20 TEPC1035 is an antidextran with specificity different from those of other murine myeloma antidextrans and from human antidextrans reported previously and its combining site size is at least as large as a trisaccharide. The binding constant of methyl alpha-D-glucoside (7.2 X 10(2)) was 73% of that of panose and comparable to that of myeloma protein W3129 (9.4 X 10(2)) with a cavity-type site and 600 times lower (1.6 X 10(0)) for QUPC52 with a groove type site, indicating that the terminal nonreducing residue is held in a cavity. Inhibition data with various alpha(1 leads to 4)-linked oligosaccharides also indicate that the internal portions of these inhibitors may react directly with a portion of the combining site. These findings suggest that myeloma antidextran CAL20 TEPC1035 has a partial cavity-type combining site in which the terminal nonreducing dGlc alpha(1 leads to 6) moiety is held in a cavity with the other two sugars forming a groove. However, oligosaccharides with one or more alternating [leads to 3DGlc alpha(1 leads to 6)DGlc alpha(1 leads to 3)DGlcl leads to] units with and without terminal nonreducing DGlc alpha(1 leads to 6) or DGLc alpha(1 leads to 3) side chains remain to be tested to determine whether structures known to be present in the three dextrans which precipitate CAL20 TEPC1035 may not prove to be more active than panose.

scholarly journals Identification and glucan-binding properties of a new carbohydrate-binding module family

2002 ◽  
Vol 361 (1) ◽  
pp. 35 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
Mazyar GHAFFARI ◽  
R. Antony J. WARREN ◽  
Douglas G. KILBURN
Keyword(s):  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Binding Properties

scholarly journals A novel mechanism of xylan binding by a lectin-like module from Streptomyces lividans xylanase 10A

2000 ◽  
Vol 350 (3) ◽  
pp. 933-941 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
Peter TOMME ◽  
Emily A. AMANDORON ◽  
Douglas G. KILBURN
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Streptomyces Lividans ◽  
Association Constants ◽  
Carbohydrate Binding ◽  
Toxin B ◽  
Ricin Toxin ◽  
Functional Sites ◽  
Directed Mutation ◽  
Single Binding Site

The C-terminal module of xylanase 10A from Streptomyces lividans is a family 13 carbohydrate-binding module (CBM13). CBM13 binds mono- and oligo-saccharides with association constants of ≈ 1×102 M-1−1×103 M-1. It appears to be specific only for pyranose sugars. CBM13 binds insoluble and soluble xylan, holocellulose, pachyman, lichenan, arabinogalactan and laminarin. The association constant for binding to soluble xylan is (6.2±0.6)×103/mol of xylan polymer. Site-directed mutation indicates the involvement of three functional sites on CBM13 in binding to soluble xylan. The sites are similar in sequence, and are predicted to have similar structures, to the α, β and γ sites of ricin toxin B-chain, which is also in family 13. The affinity of a single binding site on CBM13 for soluble xylan is only ≈ (0.5±0.1)×103/mol of xylan. The binding of CBM13 to soluble xylan involves additive and co-operative interactions between the three binding sites. This mechanism of binding has not previously been reported for CBMs binding polysaccharides. CBM13 is the first bacterial module from family 13 to be described in detail.

scholarly journals Carbohydrate binding properties of complex-type oligosaccharides on immobilized Datura stramonium lectin.

1987 ◽  
Vol 262 (4) ◽  
pp. 1602-1607 ◽  
Author(s):  
K Yamashita ◽  
K Totani ◽  
T Ohkura ◽  
S Takasaki ◽  
I J Goldstein ◽  
...  
Keyword(s):  
Datura Stramonium ◽  
Carbohydrate Binding ◽  
Complex Type ◽  
Binding Properties
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