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J. Biol. Chem., Vol. 278, Issue 25, 23027-23035, June 20, 2003

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Identification of a Lipoprotein Lipase Cofactor-binding Site by Chemical Cross-linking and Transfer of Apolipoprotein C-II-responsive Lipolysis from Lipoprotein Lipase to Hepatic Lipase^*

Trina L. McIlhargey  , Yingying Yang , Howard Wong ¶ || ** and John S. Hill  

From the University of British Columbia McDonald Research Laboratories/iCAPTUR⁴E Centre, Department of Pathology and Laboratory Medicine, St. Paul's Hospital, and the University of British Columbia, Vancouver, British Columbia V6Z 1Y6, Canada, the ^¶Department of Veterans Affairs, Greater Los Angeles Healthcare System, Los Angeles, California 90073, and the ^||Department of Medicine, University of California, Los Angeles, California 90095

To localize the regions of lipoprotein lipase (LPL) that are responsive to activation by apoC-II, an apoC-II peptide fragment was cross-linked to bovine LPL. Following chemical hydrolysis and peptide separation, a specific fragment of LPL (residues 65–86) was identified to interact with apoC-II. The fragment contains regions of amino acid sequence dissimilarity compared with hepatic lipase (HL), a member of the same gene family that is not responsive to apoC-II. Using site-directed mutagenesis, two sets of chimeras were created in which the two regions of human LPL (residues 65–68 and 73–79) were exchanged with the corresponding human HL sequences. The chimeras consisted of an HL backbone with the suspected LPL regions replacing the corresponding HL sequences either individually (HL_{LPL-(65–68)} and HL_{LPL-(73–79)}) or together (HL_LPLD). Similarly, LPL chimeras were created in which the candidate regions were replaced with the corresponding HL sequences (LPL_HL-(77–80), LPL_HL-(85–91), and LPL_HLD). Using a synthetic triolein substrate, the lipase activity of the purified enzymes was measured in the presence and absence of apoC-II. Addition of apoC-II to HL_{LPL-(65–68)} and HL_{LPL-(73–79)} did not significantly alter their enzyme activity. However, the activity of HL_LPLD increased 5-fold in the presence of apoC-II compared with an increase in native LPL activity of 11-fold. Addition of apoC-II to LPL_HL-(77–80) resulted in 10-fold activation, whereas only 6- and 4-fold activation of enzyme activity was observed in LPL_HL-(85–91) and LPL_HLD, respectively. In summary, our results have identified 11 amino acid residues in the N-terminal domain of LPL (residues 65–68 and 73–79) that appear to act cooperatively to enable substantial activation of human LPL by apoC-II.

Received for publication, January 10, 2003 , and in revised form, February 19, 2003.

^* This work was supported in part by the Heart and Stroke Foundation of British Columbia and Yukon. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a research traineeship from the Heart and Stroke Foundation of British Columbia and Yukon.

^** Supported by the Veterans Affairs Merit Review and National Institutes of Health Grant HL28481.

Scholar of the Heart and Stroke Foundation of Canada and the Michael Smith Foundation for Heath Research. To whom correspondence should be addressed: St. Paul's Hospital, Healthy Heart Program, 1081 Burrard St., Vancouver, BC V6Z 1Y6, Canada. Tel.: 604-806-8616; Fax: 604-806-8590; E-mail: jshill{at}interchange.ubc.ca.

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