Functional Analyses of Human Apolipoprotein CII by Site-directed
Mutagenesis
IDENTIFICATION OF RESIDUES IMPORTANT FOR ACTIVATION OF
LIPOPROTEIN LIPASE*
Yan
Shen
§,
Aivar
Lookene¶,
Solveig
Nilsson, and
Gunilla
Olivecrona
From the Department of Medical Biosciences, Umeå
University, Umeå SE-90 187, Sweden and ¶ National Institute of
Chemical Physics and Biophysics, Tallinn, Estonia
Apolipoprotein CII (apoCII) activates lipoprotein
lipase (LPL). Seven residues, located on one face of a model 
-helix
spanning residues 59-75, are fully conserved in apoCII from ten
different animal species. We have mutated these residues one by one.
Substitution of Ala59 by glycine, or
Thr62 and Gly65 by alanine did not
change the activation, indicating that these residues are outside the
LPL-binding site. Replacement of Tyr63, Ile66,
Asp69, or Gln70 by alanine lowered the affinity
for LPL and the catalytic activity of the LPL·apoCII complex.
For each residue several additional replacements were made. Most
mutants retained some activating ability, but replacement of
Tyr63 by phenylalanine or tryptophan and Gln70
by glutamate caused almost complete loss of activity. All mutants bound
to liposomes with similar affinity as wild-type apoCII, and they also
bound with similar affinity to LPL in the absence of hydrolyzable
lipids. However, the inactive mutants did not compete with wild-type
apoCII in the activation assay. Therefore, we conclude that the
productive apoCII·LPL interaction may be dependent on substrate
molecules. In summary, our data demonstrate that residues 63, 66, 69, and 70 are of special importance for the function of apoCII, but no
single amino acid residue is absolutely crucial.
*
This work was supported by the Swedish Medical Research
Council (Grants 727 and 12203), by the Swedish Royal Academy of
Sciences, by the Estonian Science Foundation (Grant 4925), and by
Donation Funds at the Medical Faculty, Umeå University.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
