Streptomyces lividans glycosylates the linker region of a beta-1,4-glycanase from Cellulomonas fimi.
The beta-1,4-glycanase Cex of the gram-positive bacterium Cellulomonas fimi is a glycoprotein comprising a C-terminal cellulose-binding domain connected to an N-terminal catalytic domain by a linker containing only prolyl and threonyl (PT) residues. Cex is also glycosylated by Streptomyces lividans. The glycosylation of Cex produced in both C. fimi and S. lividans protects the enzyme from proteolysis. When the gene fragments encoding the cellulose-binding domain of Cex (CBDCex), the PT linker plus CBDCex (PT-CBDCex), and the catalytic domain plus CBDCex of Cex were expressed in S. lividans, only PT-CBDCex was glycosylated. Therefore, all the glycans must be O linked because only the PT linker was glycosylated. A glycosylated form and a nonglycosylated form of PT-CBDCex were produced by S. lividans. The glycosylated form of PT-CBDCex was heterogeneous; its average carbohydrate content was approximately 10 mol of D-mannose equivalents per mol of protein, but the glycans contained from 4 to 12 alpha-D-mannosyl and alpha-D-galactosyl residues. Glycosylated Cex from S. lividans was also heterogeneous. The presence of glycans on PT-CBDCex increased its affinity for bacterial microcrystalline cellulose. The location of glycosylation only on the linker region of Cex correlates with the properties conferred on the enzyme by the glycans.
Ong
E
E
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Kilburn
D G
DG
Miller
R C
RC
Warren
R A
RA
D000327:Adsorption; D000595:Amino Acid Sequence; D002482:Cellulose / Q000378:metabolism; D005690:Galactose / Q000032:analysis; D005798:Genes, Bacterial / Q000235:genetics; D044643:Glucan 1,4-beta-Glucosidase; D006093:Gram-Positive Asporogenous Rods / Q000201:enzymology* / Q000235:genetics; D008358:Mannose / Q000032:analysis; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis; D011392:Proline / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D011134:Polysaccharides; D011994:Recombinant Proteins; D013912:Threonine; D002482:Cellulose; D011392:Proline; D001617:beta-Glucosidase; D044643:Glucan 1,4-beta-Glucosidase; D008358:Mannose; D005690:Galactose