Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms.
Human alpha1-antitrypsin (A1PI) is a well-known glycoprotein in human plasma important for the protection of tissues from proteolytic enzymes. The three N-glycosylation sites of A1PI contain diantennary N-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for A1PI. Here we present an approach to characterize A1PI isoforms from human plasma and its PTMs by LC-ESI-MS and LC-ESI-MS/MS of peptides obtained by proteolytic digestion. The single cysteine residue of A1PI formed a disulfide bridge with free cysteine. The variability of the number of antennae and hence sialic acids on glycosylation site N107, which even contained minute amounts of tetraantennary structures, emerged as a major cause for the IEF pattern of A1PI. Only negligible amounts of triantennary structures were identified attached to N70, and exclusively diantennary structures were present on site N271 in each of the isoforms analyzed. Exoglycosidase digests revealed alpha2,6-linked neuraminic acids on diantennary N-glycans, and triantennary contained additionally one single alpha2,3-neuraminic acid per N-glycan, which, together with a fucose, formed a sialyl Lewis X determinant on the beta1,4-linked N-acetylglucosamine, as shown by 2-D-HPLC of pyridylaminated asialoglycans. Fucosylation of diantennary structures was marginal and of the core alpha1,6 type.
Kolarich
Daniel
D
Department of Chemistry, Biochemistry Division, University of Natural Resources and Applied Life Sciences (BOKU), Vienna, Austria. daniel.kolarich@boku.ac.at
Weber
Alfred
A
Turecek
Peter L
PL
Schwarz
Hans-Peter
HP
Altmann
Friedrich
F
D002236:Carbohydrate Conformation; D003545:Cysteine / Q000737:chemistry*; D005643:Fucose / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D020033:Protein Isoforms / Q000737:chemistry; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D000515:alpha 1-Antitrypsin / Q000737:chemistry*
D010446:Peptide Fragments; D011134:Polysaccharides; D020033:Protein Isoforms; D000515:alpha 1-Antitrypsin; D005643:Fucose; D003545:Cysteine