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Subdomain chimeras of hepatic lipase and lipoprotein lipase - localization of heparin and cofactor binding

Academic Article
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Overview

authors

  • Hill, J. S.
  • Yang, D.
  • Nikazy, J.
  • Curtiss, Linda
  • Sparrow, J. T.
  • Wong, H.

publication date

  • November 1998

journal

  • Journal of Biological Chemistry  Journal

abstract

  • To specify and localize carboxyl-terminal domain functions of human hepatic lipase (HL) and human lipoprotein lipase (LPL), two subdomain chimeras were created in which portions of the carboxyl-terminal domain were exchanged between the two lipases. The first chimera (HL-LPLC1) was composed of residues 1-344 of human HL, residues 331-388 of human LPL, and residues 415-476 of human HL. The second chimera (HL-LPLC2) consisted of just two segments, residues 1-414 of human HL and residues 389-448 of human LPL. These chimeric constructs effectively divided the HL C-terminal domain into halves, with corresponding LPL sequences either in the first or second portion of that domain. Both chimeras were lipolytically active and hydrolyzed triolein emulsions to a similar extent compared with native HL and LPL. Heparin-Sepharose chromatography demonstrated that HL-LPLC1 and HL-LPLC2 eluted at 0.80 and 1.3 M NaCl, respectively, elution positions that corresponded to native HL and LPL. Hence, substitution of LPL sequences into the HL carboxyl-terminal domain resulted in the production of functional lipases, but with distinct heparin binding properties. In addition, HL-LPLC2 trioleinase activity was responsive to apoC-II activation, although the -fold stimulation was less than that observed with native LPL. Moreover, an apoC-II fragment (residues 44-79) was specifically cross-linked to LPL and HL-LPLC2, but not to HL or HL-LPLC1. Finally, both chimeras hydrolyzed phospholipid with a specific activity similar to that of HL, which was unaffected by the presence of apoC-II. These findings indicated that in addition to a region found within the amino-terminal domain of LPL, apoC-II also interacted with the last half of the carboxyl-terminal domain (residues 389-448) to achieve maximal lipolytic activation. In addition, the relative heparin affinity of HL and LPL was determined by the final 60 carboxyl-terminal residues of each enzyme.

subject areas

  • Apolipoprotein C-II
  • Apolipoproteins C
  • Binding Sites
  • Chromatography, Affinity
  • Cross-Linking Reagents
  • Enzyme Activation
  • Heparin
  • Humans
  • Lipase
  • Lipolysis
  • Lipoprotein Lipase
  • Recombinant Fusion Proteins
  • Structure-Activity Relationship
  • Triolein
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.273.47.30979

PubMed ID

  • 9812994
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Additional Document Info

start page

  • 30979

end page

  • 30984

volume

  • 273

issue

  • 47

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