Separation of yeast asparagine-linked oligosaccharides by high-performance anion-exchange chromatography.
Oligosaccharides obtained from Saccharomyces cerevisiae mannoproteins by digestion with endo-N-acetyl-beta-D-glucosaminidase H were fractionated by anion-exchange chromatography, by elution with 50-100mM NaOH without or with a sodium-acetate gradient, and detected with a pulsed amperometric detector (PAD). The elution times of homologous oligosaccharides fell on a straight line having a slope characteristic of the structural type. The response of the PAD detector per mole of oligosaccharide increased about 2-fold going from Man3GlcNAc to Man13GlcNAc, and appeared to depend primarily on the oxidation of the reducing-end N-acetylglucosamine unit common to all the oligosaccharides. The digestion of a Man10GlcNAc with jack-bean alpha-mannosidase was monitored by injecting portions of the crude reaction mixture, and the intermediates were characterized by their elution positions and n.m.r. spectra in the anomeric proton region. One commercial jack-bean alpha-mannosidase preparation contained a novel endolytic activity that released N-acetylglucosamine from the reducing ends of the oligosaccharides and was shown to convert P----6 alpha Man----6 alpha Man----6 beta Man----4 alpha beta GlcNAc to P----6 alpha Man----6 alpha Man----6 alpha beta Man plus free N-acetylglucosamine. Another commercial jack-bean alpha-mannosidase converted the Man10GlcNAc to a Man3GlcNAc having the structure alpha Man----6 beta Man----4 alpha beta GlcNAc, [formula: see text] whereas the Oerskovia sp. alpha-mannosidase converted the same oligosaccharide to a Man4GlcNAc having the structure alpha Man----6 alpha Man----6 beta Man----4 alpha beta GlcNAc. [formula: see text]
Hernandez
L M
LM
Department of Biochemistry, University of California, Berkeley 94720.
Ballou
L
L
Ballou
C E
CE
D000469:Alkaline Phosphatase; D001216:Asparagine / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002268:Carboxypeptidases / Q000737:chemistry; D043402:Cathepsin A; D002852:Chromatography, Ion Exchange / Q000379:methods; D004952:Esters; D008361:Mannosidases; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000032:analysis*; D029701:Saccharomyces cerevisiae Proteins
D004952:Esters; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D001216:Asparagine; D000469:Alkaline Phosphatase; D008361:Mannosidases; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase