Isolation and characterization of human milk bile salt-activated lipase C-tail fragment.
Glycosylation positions and oligosaccharide characteristics in the proline-rich, mucin-like, C-terminal region (C-tail) of human milk bile salt-activated lipase (BAL) were studied in order to assess the possible physiological functions of this region. A large-scale purification method has been devised to purify the C-tail fragment from human milk BAL. Chymotryptic, tryptic, and cyanogen bromide cleavages of partially purified BAL and subsequent molecular sieve chromatography yielded 20-30 mg of C-tail fragment from 1 L of human milk. The N-terminal sequence and amino acid composition of the purified C-tail fragment establish that it is derived from residues 528-712 of the enzyme. The O-glycosylated carbohydrates of the C-tail fragment contain fucose, galactose, glucosamine, galactosamine, and neuraminic acid in a molar ratio of 1:3:2:1:0.3, respectively. beta-Elimination reaction revealed that nine threonine residues and less than one serine residue were glycosylated. Edman degradation of C-tail fragment and its pronase subfragment suggest a number of glycosylation sites which are flanked by a consensus motif of PVPP. We suggest that this motif may serve as a signal for O-glycosylation in the C-tail region of BAL. Immunochemical studies indicated that the oligosaccharide chains in the C-tail region of BAL contain Lewis x and Lewis a antigens and, less prominently, sialyl Lewis x and sialyl Lewis a antigens. C-tail fragment was also found to bind jacalin lectin. These observations suggest the possibility that the C-tail region may contribute to adhesive activity in the physiological function of BAL.
Wang
C S
CS
Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Dashti
A
A
Jackson
K W
KW
Yeh
J C
JC
Cummings
R D
RD
Tang
J
J
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism; D003488:Cyanogen Bromide; D004789:Enzyme Activation / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification*; D008895:Milk, Human / Q000201:enzymology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D002787:Sterol Esterase; D014357:Trypsin / Q000378:metabolism
D002241:Carbohydrates; D037102:Lectins; D009844:Oligosaccharides; D010446:Peptide Fragments; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase; D008049:Lipase; D002918:Chymotrypsin; D014357:Trypsin; D003488:Cyanogen Bromide