Recombinant human milk bile salt-stimulated lipase. Catalytic activity is retained in the absence of glycosylation and the unique proline-rich repeats.
Human milk bile salt-stimulated lipase ensures efficient utilization of triacylglycerol by breast-fed infants. Cloning and sequencing of cDNA have revealed that the peptide chain consists of 722 amino acid residues showing only little homology to typical lipases. The sequence is identical to that of pancreatic carboxylic-ester hydrolase. The COOH-terminal part contains 16 proline-rich repeats of 11 residues with O-linked carbohydrate. The only N-linked sugar chain is situated close to the active-site serine. Using C127 cells and a bovine papilloma virus vector, high and stable expression of full-length lipase and of several variants, obtained by site-directed mutagenesis, was achieved. The produced proteins were purified and further characterized. Variants lacking all, or all but two, repeats were active with similar specific activity and the same bile salt dependence as the native milk enzyme. Changing the asparagine necessary for N-glycosylation gave the same principal results. Active recombinant full-length lipase was also produced in a bacterial system. We conclude that neither glycosylation (N- or O-linked) nor the proline-rich repeats are essential for catalytic activity or bile salt activation of human milk bile salt-stimulated lipase.
Hansson
L
L
Symbicom AB, Umeå, Sweden.
Bläckberg
L
L
Edlund
M
M
Lundberg
L
L
Strömqvist
M
M
Hernell
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011392:Proline / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002787:Sterol Esterase
D012333:RNA, Messenger; D011994:Recombinant Proteins; D011392:Proline; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; D008049:Lipase