Biochemical and biophysical characterization of human recombinant lecithin: cholesterol acyltransferase.
We established a Chinese hamster ovary cell line that constitutively expresses up to 5 mg/L of human recombinant lecithin: cholesterol acyltransferase (rLCAT). We purified the rLCAT to > 96% purity, and characterized it along with plasma LCAT (pLCAT) biochemically and biophysically. The recombinant enzyme is more heavily glycosylated and more heterogeneous in its carbohydrate content than the plasma enzyme, as revealed by differences in molecular weight and pI isoforms, determined by mass spectrometry and isoelectric focusing. Recombinant LCAT is half as active enzymatically as pLCAT. The difference in activity is due to differences in the catalytic rates rather than in the apparent K(m) values, suggesting that the binding of the rLCAT to interfaces is not altered by its different glycosylation pattern. Despite these differences, rLCAT has essentially the same intrinsic tryptophan fluorescence emission spectrum and far-UV CD spectrum as pLCAT, indicating that the tertiary and secondary structures of both enzyme forms are very similar. Both enzyme forms have a propensity to self-associate, and their multimers appear resistant to dissociation by SDS and dilution. The free energies of unfolding (delta G(H2O)) of rLCAT and pLCAT are 3.4 +/- 0.2 and 3.2 +/- 0.2 kcal/mol, respectively, as determined by guanidine hydrochloride denaturation monitored by fluorescence. These relatively low delta G(H2O) values support the notion that LCAT is capable of undergoing major conformational changes upon interaction with interfacial substrates.
Jin
L
L
Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.
Lee
Y P
YP
Jonas
A
A
D000818:Animals; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006031:Glycosylation; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011108:Polymers / Q000032:analysis; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D006146:Guanidines; D011108:Polymers; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D019791:Guanidine