Abstract
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.
Highlights
From SSymbicomA B, S-90124Umed, the Departments of Wedical Biochemistry and Biophysics and **Pediatrics, University of Umed, S-90187Umed, and lkstraHassle AB, Preclinical Research and Development, S-43183 Molndal, Sweden
Characterization of Recombinant DNA in Mammalian Cell Lines-DNA samples were prepared from the cell lines transfected with the expression vectors encoding the different BSSL variants
From cloning and sequencing of cDNAs from human milk BSSL and pancreatic carboxylic-ester hydrolase, it became evident that thesetwo lipolytic enzymes have identical polypeptide chains [11,12, 14, 15], expression of the gene is different
Summary
(Received forpublication, February 23, 1993, and in revised form, June 20, 1993). Lennart HanssonSB, LarsBlackberg n, Michael EdlundS, Lennart Lundbergll, Mats StromqvistS, and Olle Hemell**. Active-site serine [11].The BSSL sequence containsin its COOH-terminal part 16 proline-rich repeats of 11amino acid residues each. A major explanation for differences in molecular sizeand amino acid composition between corresponding enzymes from different species [16,17,18] These repeats carry most of the 15-20% carbohydrate of the protein Bjursell) [22] into plasmid pGEMEX-1 (Promega Biotec) [23] By this cloning procedure, the T7 gene 10 coding sequence was replaced by the BSSL cDNA coding for the mature protein preceded by a start codon. The BclI site was introduced via PCR-1into the BSSL sequence,without altering amino acid sequence.This was done to facilitate introduction of synthetic DNA to obtain the other variants.
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