The amino acid sequences of the carboxyl termini of human and mouse hepatic lipase influence cell surface association.
Human hepatic lipase (hHL) mainly exists cell surface bound, whereas mouse HL (mHL) circulates in the blood stream. Studies have suggested that the carboxyl terminus of HL mediates cell surface binding. We prepared recombinant hHL, mHL, and chimeric proteins (hHLmt and mHLht) in which the carboxyl terminal 70 amino acids of hHL were exchanged with the corresponding sequence from mHL. The hHL, mHL, and hHLmt proteins were catalytically active using triolein and tributyrin as substrates. In transfected cells, the majority of hHLs bound to the cell surface, with only 4% of total extracellular hHL released into heparin-free media, whereas under the same conditions, 61% of total extracellular mHLs were released. Like mHL, hHLmt showed decreased cell surface binding, with 68% of total extracellular hHLmt released. To determine the precise amino acid residues involved in cell surface binding, we prepared a truncated hHL mutant (hHL471) by deleting the carboxyl terminal five residues (KRKIR). The hHL471 also retained hydrolytic activity with triolein and tributyrin, and showed decreased cell surface binding, with 40% of total extracellular protein released into the heparin-free media. These data suggest that the determinants of cell surface binding exist within the carboxyl terminal 70 amino acids of hHL, of which the last five residues play an important role.
Brown
Robert J
RJ
Lipoprotein and Atherosclerosis Research Group, University of Ottawa, Ottawa, Ontario, Canada.
Schultz
Joshua R
JR
Ko
Kerry W S
KW
Hill
John S
JS
Ramsamy
Tanya A
TA
White
Ann L
AL
Sparks
Daniel L
DL
Yao
Zemin
Z
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D002845:Chromatography; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry*; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014280:Triglycerides / Q000737:chemistry; D014304:Triolein / Q000737:chemistry
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014280:Triglycerides; D014304:Triolein; D006493:Heparin; D012685:Sepharose; D008049:Lipase; C005830:tributyrin