Expression and characterization of recombinant human lecithin:cholesterol acyltransferase

Hill, John S.; Wang, X; Paranjape, Sulabha; Dimitrijevich, Dan; Lacko, Andras G.; Pritchard, P H

doi:10.1016/s0022-2275(20)37712-9

Cited by 34 publications

(9 citation statements)

References 32 publications

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“…Furthermore, CHO-hLCATH6 and CHO-hLCAT appeared to yield identical products after deglycosylation with NANase or PNGase, the only difference being that CHO-hLCAT6 ran slower on the gel due to the presence of the additional six histidine residues. The molecular weights and carbohydrate composition of all three LCAT species were consistent with that previously reported (9,11,16,36). Both CHO-LCAT species had characteristically broader bands by SDS-PAGE when compared to plasma LCAT and this was likely due to greater heterogeneity in the carbohydrate chains.…”

Section: Discussionsupporting

confidence: 88%

“…Isolation of CHO-hLCATH6 by cobalt metal affinity chromatography was highly efficient. The yield and purity, by SDS-PAGE, were comparable to those reported for the single-step purification of recombinant LCAT using phenyl-Sepharose (10,11). In our hands, phenyl-Sepharose chromatography yields purified LCAT, that by SDS-PAGE appears to be free of major contaminants.…”

Section: Discussionsupporting

confidence: 84%

“…Three different recombinant cell systems have been previously described for the production of LCAT. LCAT from both stably transfected CHO (9, 10) and BHK cells (11) has been shown to be very similar to plasma LCAT with the exception that they are more heavily and heterogeneously glycosylated. LCAT has also been expressed in insect cells using the baculovirus system (9,12) and although enzymatically active, contains much less carbohydrate than LCAT from mammalian sources.…”

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confidence: 99%

“…Most LCAT purifications from plasma and recombinant cell lines (9,(13)(14)(15)(16)(17) are based primarily on a combination of phenyl-Sepharose and hydroxylapatite chromatography. However, a number of investigators (10,11) have had success in purifying recombinant wild-type LCAT using a single phenyl-Sepharose step. The reliability and reproducibility of this method for LCAT mutants and chimeras remains to be determined.…”

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confidence: 99%

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Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase

Chisholm

Gebre

Parks

1999

Journal of Lipid Research

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Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catalyzes esterification of the sn -2 fatty acid of phospholipid to cholesterol. To facilitate the isolation of large quantities of LCAT and to assist in future structure-function studies, LCAT containing a carboxyterminal histidine-tag (H6) was expressed in Chinese hamster ovary cells (CHO). A high level of CHO-hLCATH6 expression ( Ϸ 15 mg L ؊ 1 ) was achieved over a 72-h period using 10 m M sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme ( Ϸ 96%) was isolated by cobalt metal affinity chromatography with an activity yield of 82 ؎ 26%. CHO-hLCATH6 and CHO-hLCAT species had identical specific activities (26 ؎ 6 and 26 ؎ 3 nmol CE formed g ؊ 1 h ؊ 1 , respectively). The enzymatic activity of CHO-hLCATH6 was stable at 4 ؇ C in excess of 60 days. Substrate saturation studies, using rHDL composed of 1-palmitoyl-2-oleoylsn -glycero-3-phosphocholine (POPC), cholesterol, and apolipoprotein A-I (80:5:1) indicated that the app K m for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at Ϸ 2 M substrate cholesterol. We conclude that carboxy-terminal histidine-tagged LCAT is a suitable replacement for both plasma LCAT and CHO-hLCAT. -Chisholm, J. W., A. K. Gebre, and J. S. Parks. Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionsupporting

confidence: 84%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase

Chisholm

Gebre

Parks

1999

Journal of Lipid Research

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“…Recombinant LCAT was produced by a baby hamster kidney (BHK) cell line, stably transfected with the LCAT cDNA, under control of the mouse metallothionein promotor (26). Stably transfected BHK cells were incubated with Dulbecco's modified Eagle medium (DMEM; GIBCO-BRL, Gaithersburg, MD) containing 10% fetal bovine serum, streptomycin (100 g/ml), and penicillin (100 units/ml).…”

Section: Production Of Wild-type Rlcat In Bhk Cellsmentioning

confidence: 99%

Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase

Roosbeek

Vanloo

Duverger³

et al. 2001

Journal of Lipid Research

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Previous studies have suggested that the helical repeat formed by residues 143-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipidprotein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V max because the apparent Michaelis-Menten constant K m values were similar for the mutant and wild-type apoA-I proteins. These data suggest that R149, R153, and R160 participate in apoA-Imediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT.

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Probing the 121–136 Domain of Lecithin:Cholesterol Acyltransferase Using Antibodies

Murray

Nair

Ayyobi

et al. 2001

Archives of Biochemistry and Biophysics

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Expression and characterization of recombinant human lecithin:cholesterol acyltransferase

Cited by 34 publications

References 32 publications

Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase

Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase

Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase

Probing the 121–136 Domain of Lecithin:Cholesterol Acyltransferase Using Antibodies

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