STRUCTURE OF HUMAN-MILK BILE-SALT ACTIVATED LIPASE

The structure and some functional sites of human milk bile salt activated lipase (BAL) were studied by cDNA cloning and chemical analysis of the enzyme. Eighteen cDNA clones of human BAL were identified from lactating human breast cDNA libraries in lambda-gt10 with antibody and synthetic oligonucleotides as probes. The sequence of four clones was sufficient to construct a 3018-bp BAL cDNA structure. This sequence codes for an open reading frame of 742 amino acid residues. There is a putative signal sequence of 20 residues which is followed by the amino-terminal sequence of BAL, and the mature BAL contains 722 amino acid residues. The cDNA sequence also contains a 678-base 5'-untranslated sequence, a 97-base 3'-untranslated region, and a 14-base poly(A) tail. The sequence of a 1.8-kbp insert of clone G10-4A differs from that of the other cDNA in that it contains a deletion of 198 bases (1966-2163) corresponding to 66 amino acid residues. By use of BAL cDNA as probe, it was found that the major molecular species of BAL mRNA in human mammary gland HBL-100 cells had a size of 2.9 kb and two minor species had sizes of 3.8 and 5.1 kb by Northern blot analyses. The deduced BAL protein structure contains in the carboxyl-terminal region 16 repeating units of 11 amino acids each. The repeating units have the basic structure Pro-Val-Pro-Pro-Thr-Gly-Asp-Ser-Gly-Ala-Pro with only minor substitutions. The amino acid sequence of human BAL is related to that of pancreatic lysophospholipase, cholesterol esterase, cholinesterase, acetylcholinesterase, and thyroglobulin. Ten of the 14 cyanogen bromide fragments of diisopropyl fluorophosphate inhibited human milk BAL were isolated, determined for N-terminal sequences, analyzed for amino sugars, and tested for some functional properties. These chemical studies established that the active site of human milk BAL is located at serine-194, the N-glycosylation site is present at asparagine-187, the O-glycosylation region is in the 16 repeating units near the C-terminus, and the heparin binding domain is in the N-terminal region. We have also determined the location of disulfide bridges as Cys64-Cys80 and Cys246-Cys257. The cyanogen bromide cleavage and the partial sequencing of CNBr peptides also confirmed the location of methionines in the polypeptide chain as well as the deduced cDNA sequence of BAL.