ABSTRACT
Human pancreatic elastase 1 (E1) is a glycoprotein containing two potential N-glycosylation sites, one of which carries a carbohydrate moiety [Wendorf, Geyer, Sziegoleit & Linder (1989) FEBS Lett. 249, 275-278]. In order to study its glycosylation, glycoprotein isolated from post-mortem pancreas tissue of 75 donors was digested with trypsin. Oligosaccharides were liberated from resulting glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glycosaminyl)-asparagine amidase F, radiolabelled by reduction with KB3H4 and separated by h.p.l.c. and gel filtration. Major oligosaccharide alditol fractions, representing 67.8 mol% of total glycans, were characterized by methylation analysis and sequential degradation with exoglycosidases. The results revealed that about two-fifths of the partially truncated, mainly biantennary, complex-type glycans found comprised blood group A, B, Lea (or X), difucosyl A or difucosyl B determinants, which could be assigned to lactosamine antennae linked to Man(alpha 1-3)- residues of the sugar chains.
Subject(s)
Oligosaccharides/chemistry , Pancreas/enzymology , Pancreatic Elastase/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Glycopeptides/metabolism , Glycosylation , Humans , Hydrogen-Ion Concentration , Methylation , Molecular Sequence Data , Oligosaccharides/metabolism , Pancreatic Elastase/isolation & purification , Sugar Alcohols/chemistry , Trypsin/chemistryABSTRACT
A series of high-mannose and complex type glycoprotein-N-glycans was subjected to high-pH anion-exchange chromatography. The results revealed that this method represents a useful tool for analytical characterization of single oligosaccharides as well as preparative separation of complex mixtures of carbohydrate side-chains. On the other hand, it became evident that in several cases a combination of different chromatographic techniques is required for efficient separation of individual oligosaccharide species.
Subject(s)
Glycoproteins/isolation & purification , Polysaccharides/isolation & purification , Chromatography, Ion Exchange , Hexosaminidases , Hydrogen-Ion Concentration , Methylation , Oligosaccharides/isolation & purificationABSTRACT
Crystalline elastase 1 from human pancreas was digested with trypsin. Two peptides, containing the potential N-glycosylation sites at Asn-86 and Asn-125, were isolated and analyzed by amino acid analysis, sequencing and carbohydrate component analysis. The results demonstrate that only Asn-86 is glycosylated.
Subject(s)
Pancreatic Elastase/metabolism , Amino Acid Sequence , Amino Acids/analysis , Chromatography, Gel , Glycopeptides/isolation & purification , Glycosylation , Humans , Hydrolysis , Molecular Sequence DataABSTRACT
This paper is a continuation of our study of various animal pancreatic enzymes which are related to human pancreatic elastase 1 (Sziegoleit, A. & Linder, D. (1986) Biol. Chem. Hoppe-Seyler, 367, 527-531). The isolation and immunological analysis of the related protein from bovine pancreas disclosed that the third subunit of the procarboxypeptidase A complex is the antibody-binding component. The similarity of this subunit to elastase 1 is affirmed by comparison of their primary structures. While the complete amino-acid sequence of bovine subunit III recently has been published (Venot, N., Sciaky, M., Puigserver, A., Desnuelle, P. & Laurent, G. (1986) Eur. J. Biochem. 157, 91-99), we here present the amino-acid sequence of the carboxy-terminal tryptic peptide of human pancreatic elastase 1 showing a high degree of homology.
