Mass spectrometric characterization and glycosylation profile of bovine pancreatic bile salt-activated lipase.
We developed a procedure for the large scale isolation of bovine bile salt-activated lipase (BAL) for its crystallization [Wang, X., et al. (1997) Structure 5, 1209-1218] and also carried out a study on the molecule's glycosylation profile for a better deduction of the structure of the enzyme. Mass spectrometric analysis of the CNBr-generated peptides indicated that only one (Asn-361) of the two potential N-glycosylation sites (Asn-187 and Asn-361) with NXT motif is glycosylated. The analysis of the isolated CNBr peptide containing Asn-361 showed that it existed in three glycoforms in a ratio of 1.0:2.8:1.0, with oligosaccharide moieties weighing 1900.1, 2045.2, and 2336.4 Da, respectively. The major oligosaccharide chain contained mannose: galactose:N-acetylglucosamine:fucose:sialic acid in a molar ratio of 2:2:4:2:1. It was also determined that the potential O-glycosylated peptide (CB13) is not O-glycosylated and, in addition, it was found that there was microheterogeneity in the C-terminus of the isolated bovine BAL. The results obtained from this mass spectrometric study combined with the X-ray crystallographic studies provide more precise structural information on BAL. The procedure described here for the mass spectrometric analysis of CNBr-generated peptides also has general applicability for analysis of the glycosylation profile of glycoproteins and the C-terminal peptide structure of proteins.
Wang
C S
CS
Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City 73014, USA.
Jackson
K W
KW
Dashti
A
A
Downs
D
D
Zhang
X
X
Tang
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D002787:Sterol Esterase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; D014357:Trypsin; D003488:Cyanogen Bromide