Tài liệu Báo cáo Y học: Molecular modeling of the dimeric structure of human lipoprotein lipase and

Molecular modeling of the dimeric structure of human lipoprotein

lipase and functional studies of the carboxyl-terminal domain

Yoko Kobayashi, Toshiaki Nakajima and Ituro Inoue

Division of Genetic Diagnosis, Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Lipoprotein lipase (LPL) plays a key role in lipid metabo￾lism. Molecular modeling of dimeric LPL was carried out

using INSIGHT II based upon the crystal structures of human,

porcine, and horse pancreatic lipase. The dimeric model

reveals a saddle-shaped structure and the key heparin￾binding residues in the amino-terminal domain located on

the top of this saddle. The models of two dimeric

conformations – a closed, inactive form and an open, active

form – differ with respect to how surface-loop positions

affect substrate access to the catalytic site. In the closed form,

the surface loop covers the catalytic site, which becomes

inaccessible to solvent. Large conformational changes in the

open form, especially in the loop and carboxyl-terminal

domain, allow substrate access to the active site. To dissect

the structure–function relationships of the LPL carboxyl￾terminal domain, several residues predicted by the model

structure to be essential for the functions of heparin binding

and substrate recognition were mutagenized. Arg405 plays

an important role in heparin binding in the active dimer.

Lys413/Lys414 or Lys414 regulates heparin affinity in both

monomeric and dimeric forms. To evaluate the prediction

that LPL forms a homodimer in a
head-to-tail orientation,

two inactive LPL mutants – a catalytic site mutant (S132T)

and a substrate-recognition mutant (W390A/W393A/

W394A) – were cotransfected into COS7 cells. Lipase

activity could be recovered only when heterodimerization

occurred in a head-to-tail orientation. After cotransfection,

50% of the wild-type lipase activity was recovered, indica￾ting that lipase activity is determined by the interaction

between the catalytic site on one subunit and the substrate￾recognition site on the other.

Keywords: lipoprotein lipase; dimeric model structure;

heparin binding; substrate recognition; catalytic activity.

Lipoprotein lipase (LPL) belongs to a mammalian lipase

family that includes pancreatic lipase (PL), hepatic lipase

(HL), gastric lipase, and endothelial lipase [1,2]. The

primary function of LPL is triglyceride hydrolysis in

triglyceride-rich lipoproteins, such as chylomicron and very

low density lipoprotein (VLDL) particles, which are

converted to remnants. LPL is secreted from a variety of

tissues, such as adipocyte, macrophage, and muscle cells,

and is bound to the capillary bed of endothelium via cellular

surface heparan sulfate proteoglycans (HSPG), a function

reflected in LPL’s strong affinity for heparin. LPL defici￾encies in humans are manifested as severe hypertriglyceri￾demia [3–5] and arteriosclerosis [6]. Genetically engineered

mice lacking LPL also exhibit hypertriglyceridemia. In

addition to lipolytic activity, LPL functions as a ligand for

lipoprotein receptors, such as low density lipoprotein (LDL)

receptor, LDL receptor related protein (LRP), GP330/

LRP-2, and VLDL receptor [7–11].

A model structure of LPL had previously been construc￾ted, based on the crystal structure of human PL as a

template [12]. The model structure exhibited two domains –

a large N-terminal domain (1–312 amino acid residues) and

a small C-terminal domain (313–448 residues). The

sequences of PL and LPL are identical at 31% of their

residues in the N-terminal domain (40% similarity) and are

28% identical in the C-terminal domain (38% similarity).

The catalytic efficiency and heparin-binding functions of

the N-terminal domain have been extensively studied

[13,14]. A chimeric enzyme with the N-terminal domain of

LPL and the C-terminal domain of HL (LPL/HL) exhibited

the characteristic catalytic activity of LPL, as well as other

LPL-specific functions, such as activation by ApoC-II

and inhibition by NaCl [15]. Horse PL [16], human PL [17],

and complexes of human PL and procolipase [18,19] have

been crystallized. These studies demonstrated that the active

site in the N-terminal domain has two conformations – an

active, open conformation and an inactive, closed confor￾mation [18]. A surface loop functions as a lid and governs

the interaction of the lipid substrate with the enzyme’s

catalytic site [20]. On the protein surface at a site opposite to

the lid, occurs a cluster of basic amino acids (Arg279,

Lys280, Arg282) that constitutes a high-affinity, heparin￾binding site [14].

The function of the C-terminal domain has also been

addressed with a chimeric enzyme (LPL/HL), which

exhibits an affinity for heparin similar to that of native

LPL [21], suggesting that the major heparin-binding site

occurs in LPL’s N-terminal domain. Recently, however,

several lines of evidence have demonstrated that the

Correspondence to I. Inoue, Division of Genetic Diagnosis, Institute

of Medical Science, The University of Tokyo, Shirokanedai 4-6-1,

Minato-ku, Tokyo 108-8639, Japan.

Fax: + 81 3 5449 5764, Tel.: + 81 3 5449 5325,

E-mail: [email protected]

Abbreviations: LPL, lipoprotein lipase; PL, pancreatic lipase;

HL, hepatic lipase; VLDL, very low density lipoprotein; HSPG,

heparan sulfate proteoglycans; LDL, low density lipoprotein; LRP,

LDL receptor related protein; DMEM, Dulbecco’s modified Eagle’s

medium; ADIFAB, acrylodated intestinal fatty acid binding protein.

(Received 17 May 2002, revised 26 July 2002, accepted 13 August 2002)

Eur. J. Biochem. 269, 4701–4710 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03179.x