Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase

Ghosh, Debashis; Wawrzak, Z.; Плетнев, В. З.; Li, Naiyin; Kaiser, Rudolf; Pangborn, W.; Jörnvall, Hans; Erman, Mary; Duax, William L.

doi:10.1016/s0969-2126(01)00158-7

Cited by 116 publications

(111 citation statements)

References 34 publications

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“…Interestingly, a similar phenomenon is observed with the distantly related fungal lipase from Candida cylindracea [19] which exists in equilibrium with dimeric and monomeric forms [20]. Recent structural analysis has revealed a dimeric association of monomers in which the two active-site cavities face each other, shielding hydrophobic surfaces from the aqueous environment [21].…”

Section: Role Of the C-terminal Glycosylated Domain In Thementioning

confidence: 54%

Bile salt activation of human cholesterol esterase does not require protein dimerisation

Loomes

Senior

1997

FEBS Letters

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Human milk cholesterol esterase (bile salt-activated lipase) plays a role in the dietary uptake of triacylglyceride and cholesteryl ester. The activities toward these substrates are mediated through a unique bile salt-activated mechanism. Previously, it has been proposed that a necessary step in this process is prior protein dimerisation in the presence of primary bile salts. In this study, we addressed the role of protein dimerisation by investigating bile salt interactions on full length and truncated recombinant forms, as analysed by size exclusion chromatography and concanavalin A Sepharose binding experiments. The present findings demonstrate that protein dimerisation is not an obligatory component of the bile salt-activated pathway. A new functional role for the glycosylated C-terminal domain in cholesterol esterase is also demonstrated in the prevention of non-specific hydrophobic interactions.

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Section: Role Of the C-terminal Glycosylated Domain In Thementioning

confidence: 54%

Bile salt activation of human cholesterol esterase does not require protein dimerisation

Loomes

Senior

1997

FEBS Letters

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“…Fatty acid binding to cutinase (Longhi et al, 1996), to Rhizomucor miehei lipase (Vase1 et al, 1993;Norin et al, 1994), and to human pancreatic lipase (Van Tilbeurgh et al, 1992), involves similar interactions with a residue N-terminal of helix a4-5. The accommodation of the cholesterol nucleus in LCAT can be predicted from the structural and functional homology between LCAT and Candida cylindracea cholesterol esterase (Ghosh et al, 1995). as both enzymes can function as an acyl transferase and as an esterase.…”

Section: Model Building and Biological Implicationsmentioning

confidence: 99%

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling

et al. 1998

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The enzyme cholesterol lecithin acyl transferase (LCAT) shares the Ser/Asp‐Glu/His triad with lipases, esterases and proteases, but the low level of sequence homology between LCAT and these enzymes did not allow for the LCAT fold to be identified yet. We, therefore, relied upon structural homology calculations using threading methods based on alignment of the sequence against a library of solved three‐dimensional protein structures, for prediction of the LCAT fold. We propose that LCAT, like lipases, belongs to the α/β hydrolase fold family, and that the central domain of LCAT consists of seven conserved parallel beta‐strands connected by four α‐helices and separated by loops. We used the conserved features of this protein fold for the prediction of functional domains in LCAT, and carried out site‐directed mutagenesis for the localization of the active site residues. The wild‐type enzyme and mutants were expressed in Cos‐1 cells. LCAT mass was measured by ELISA, and enzymatic activity was measured on recombinant HDL, on LDL and on a monomelic substrate. We identified D345 and H377 as the catalytic residues of LCAT, together with F103 and L182 as the oxyanion hole residues. In analogy with lipases, we further propose that a potential “lid” domain at residues 50‐74 of LCAT might be involved in the enzyme‐substrate interaction. Molecular modeling of human LCAT was carried out using human pancreatic and Candida antarctica lipases as templates. The three‐dimensional model proposed here is compatible with the position of natural mutants for either LCAT deficiency or Fish‐eye disease. It enables moreover prediction of the LCAT domains involved in the interaction with the phospholipid and cholesterol substrates.

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“…Instead, it suggests that the Asn 384 glycan may have some inhibitory effect on the access or binding of the lipid substrates to the active site of LCAT. This possibility has some merit, for in an analogous enzyme, cholesterol esterase, the ␣-His helix forms part of the cholesterol binding pocket (34). However, it should be stressed that apparent K m values are not a direct measure of the binding of LCAT to the rHDL surface and should be interpreted with great caution.…”

Section: Discussionmentioning

confidence: 99%

Deletion of Specific Glycan Chains Affects Differentially the Stability, Local Structures, and Activity of Lecithin-cholesterol Acyltransferase

Kosman

Jonas

2001

Journal of Biological Chemistry

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The enzymatic and interfacial binding activity of lecithin-cholesterol acyltransferase (LCAT) is affected differentially by the location and extent of its glycosylation. Two LCAT glycosylation-deficient mutants, N84Q and N384Q, were constructed, permanently expressed in Chinese hamster ovary cells, and purified to determine the effects of deleting individual glycan chains on its stability, structure, and function. These purified mutants were studied by spectroscopic structural methods and enzymatic and binding assays to develop a molecular rationale for the relationship between LCAT glycosylation and activity. The N84Q LCAT mutant did not possess measurable enzymatic activity or interfacial binding affinity for reconstituted high-density lipoproteins. In addition, in thermal and chemical denaturation studies, N84Q LCAT was found to be significantly less stable than wild-type LCAT. The N384Q variant was initially more enzymatically active than wild-type LCAT, but gradually lost activity within months; however, it retained full interfacial binding activity. Significant changes were detected over time by circular dichroism in the ␣-helical content of N384Q LCAT and in the ␤-sheet content of N84Q LCAT, compared with wild-type LCAT. Fluorescence measurements with the probe 1-anilinonapthalene-8-sulfonate suggested an alteration of the active site cavity in both mutants. In conclusion, both mutants lost catalytic activity, N84Q shortly after purification and N384Q more gradually, and were destabilized, probably because the deletion of the glycan chains altered local structural elements near the active site cavity and/or the interfacial binding regions. Lecithin-cholesterol acyltransferase (LCAT)1 is the enzyme responsible for the generation of the majority of the cholesterol esters present in human plasma. The conversion of cholesterol to cholesterol esters is necessary to facilitate reverse cholesterol transport, the process which removes excess cholesterol and phospholipids from peripheral cells and delivers them to the liver for disposal (1). The LCAT reaction occurs primarily on the surface of high-density lipoproteins (HDL), which contains phospholipids, cholesterol, and apolipoprotein A-I (apoA-I). Although no crystal structure exists for LCAT, the study of its structure-function relationships has been facilitated by the construction of a computer model of the catalytic core of LCAT (2-6). Based on its amino acid composition alone, LCAT has an intermediate hydrophobicity between integral membrane proteins and apolipoproteins (7). One modification commonly found in plasma proteins, including LCAT, that increases their solubility and hydrophilicity is glycosylation.Four N-linked complex type glycans and two O-linked glycans are found in LCAT (8, 9) and constitute around 20% of the total enzyme mass of human plasma LCAT (10). Different cell types transfected with the human LCAT gene express the glycan structure of LCAT differently (11, 12), which leads to disparities between recombinant preparations of LCAT. Chines...

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Structure of uncomplexed and linoleate-bound Candida cylindracea cholesterol esterase

Cited by 116 publications

References 34 publications

Bile salt activation of human cholesterol esterase does not require protein dimerisation

Bile salt activation of human cholesterol esterase does not require protein dimerisation

A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling

Deletion of Specific Glycan Chains Affects Differentially the Stability, Local Structures, and Activity of Lecithin-cholesterol Acyltransferase

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