Amino acid residue 149 of lecithin:cholesterol acyltransferase determines phospholipase A2 and transacylase fatty acyl specificity.
Human LCAT prefers phosphatidylcholine (PC) with sn-1-palmitoyl-2-oleoyl PC (POPC) as substrate for cholesteryl ester synthesis, whereas rat LCAT (which is 92% similar in amino acid sequence) prefers sn-1-palmitoyl-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 were assayed for LCAT cholesterol esterification (CE) or phospholipase A2 (PLA2) activity using substrate particles containing POPC or PAPC. 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 clones produced CE activity ratios <0.3, unchanged from human LCAT. The cluster 6 clone (Y292H/W294F) had an intermediate ratio (0.6). Similar results were observed for LCAT PLA2 activity. In additional studies, position 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 PLA2 activity ratio for the hE149A construct was >1.7, similar to rat LCAT, whereas the triple mutation construct retained a ratio similar to human LCAT (<0.6). Thus, a single amino acid substitution (E149A) was sufficient to alter the fatty acyl specificity of human LCAT to that of rat LCAT, with an increase in activity toward PAPC. This is the first example of a point mutation in an enzyme with PLA2 activity that results in an increase in activity toward arachidonic acid.
Wang
J
J
Department of Comparative Medicine, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27157, USA.
Gebre
A K
AK
Anderson
R A
RA
Parks
J S
JS
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002788:Cholesterol Esters / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010215:Papio; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry*; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D051381:Rats; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013379:Substrate Specificity
D002788:Cholesterol Esters; D005971:Glutamates; D011993:Recombinant Fusion Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D010741:Phospholipases A; D054467:Phospholipases A2; D000409:Alanine