Jc. Wang et al., AMINO-ACID RESIDUE-149 OF LECITHIN-CHOLESTEROL ACYLTRANSFERASE DETERMINES PHOSPHOLIPASE A(2) AND TRANSACYLASE FATTY ACYL SPECIFICITY, The Journal of biological chemistry, 272(1), 1997, pp. 280-286
Human LCAT prefers phosphatidylcholine (PC) with sn-1-palmitoyl-2-oleo
yl PC (POPC) as substrate for cholesteryl ester synthesis, whereas rat
LCAT (which is 92% similar in amino acid sequence) prefers sn-1-palmi
toyl-2-arachidonoyl PC (PAPC), Six recombinant human LCAT cDNA clones
were constructed with unique clusters of rat sequence substitutions in
the human background spanning the region encoding amino acids 121-296
, Media from transfected COS cells expressing each of the constructs w
ere assayed for LCAT cholesterol esterification (CE) or phospholipase
A(2) (PLA(2)) activity using substrate particles containing POPC or PA
PC, The PAPC/POPC CE activity ratio of the cluster 1 construct (amino
acids 149-158) was 1.3, resembling rat LCAT, whereas cluster 2-5 clone
s produced CE activity ratios <0.3, unchanged from human LCAT, The clu
ster 6 clone (Y292H/W294F) had an intermediate ratio (0.6), Similar re
sults were observed for LCAT PLA(2) activity, In additional studies, p
osition 149 of human LCAT was changed to the rat sequence (hE149A) and
compared to a triple mutation containing the remainder of the cluster
1 changes (G151R/E154D/R158Q), CE and PLA(2) activity ratio for the h
E149A construct was >1.7, similar to rat LCAT, whereas the triple muta
tion construct retained a ratio similar to human LCAT (<0.6). Thus, a
single amino acid substitution (E149A) was sufficient to alter the fat
ty acyl specificity of human LCAT to that of rat LCAT, with an increas
e in activity toward PAPC, This is the first example of a point mutati
on in an enzyme with PLA(2) activity that results in an increase in ac
tivity toward arachidonic acid.