THE NATURE OF THE CARBOHYDRATE-PEPTIDE LINKAGE REGION IN GLYCOPROTEINS FROM THE CELLULOSOMES OF CLOSTRIDIUM-THERMOCELLUM AND BACTEROIDES-CELLULOSOLVENS

The cellulase complexes of two cellulolytic bacteria, Clostridium ther mocellum and Bacteroides cellulosolvens, were subjected to extensive P ronase digestion. Glycopeptide fractions were isolated by gel permeati on and fast protein liquid chromatography and analyzed by monosacchari de analysis, amino acid analysis, methylation analysis, and H-1 NMR sp ectroscopy. Alkaline boro-hydride-induced deglycosylation/amino acid c onversion and periodate oxidation studies on the glycopeptide fraction of the C. thermocellum cellulosome demonstrated that the earlier esta blished collection of carbohydrate moieties with 2)-[D-Galp-alpha(1--> 3)]-D-Galf-alpha(1-->2)-D-Gal (where 3-O-Me-D-GlcpNAc is 3-O-methyl-N- acetylglucopyranosamine, Galp is galactopyranose, and Galf is galactof uranose) as the major component, is O-linked to threonine via galactop yranose. Using the same approach for the glycopeptide fraction of the cellulase complex of B. cellulosolvens, it was found that the reported collection of carbohydrate moieties with >2)-D-Galf-alpha(1-->2)-[D-G alf-beta(1-->3)]-D-Gal as the major component, is O-linked mainly to t hreonine and partly to serine via galactopyranose. In both species, th e hydroxyamino-acid-bound galactopyranose residue has probably an alph a-configuration. The carbohydrate chains appear as clusters located in highly Thr/Pro-rich peptide regions of the glycoproteins. The results are consistent with the notion that the glycosylation sites are local ized in linker sequences which connect the various binding domains of the noncatalytic S1 subunit of the cellulosome.