Multimerization of Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1) and Familial Chylomicronemia from a Serine-to-Cysteine Substitution in GPIHBP1 Ly6 Domain

Wanee Plengpanich,Stephen G Young,Weerapan Khovidhunkit,André Bensadoun,Hirankorn Karnman,Michael Ploug,Henrik Gårdsvoll,Calvin S Leung,Oludotun Adeyo,Mikael Larsson,Suwanna Muanpetch,Supannika Charoen,Loren G Fong,Sathit Niramitmahapanya,Anne P Beigneux

doi:10.1074/jbc.m114.558528

Abstract

GPIHBP1, a glycosylphosphatidylinositol-anchored glycoprotein of microvascular endothelial cells, binds lipoprotein lipase (LPL) within the interstitial spaces and transports it across endothelial cells to the capillary lumen. The ability of GPIHBP1 to bind LPL depends on the Ly6 domain, a three-fingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Here, we report a patient with severe hypertriglyceridemia who was homozygous for a GPIHBP1 point mutation that converted a serine in the GPIHBP1 Ly6 domain (Ser-107) to a cysteine. Two hypertriglyceridemic siblings were homozygous for the same mutation. All three homozygotes had very low levels of LPL in the preheparin plasma. We suspected that the extra cysteine in GPIHBP1-S107C might prevent the trafficking of the protein to the cell surface, but this was not the case. However, nearly all of the GPIHBP1-S107C on the cell surface was in the form of disulfide-linked dimers and multimers, whereas wild-type GPIHBP1 was predominantly monomeric. An insect cell GPIHBP1 expression system confirmed the propensity of GPIHBP1-S107C to form disulfide-linked dimers and to form multimers. Functional studies showed that only GPIHBP1 monomers bind LPL. In keeping with that finding, there was no binding of LPL to GPIHBP1-S107C in either cell-based or cell-free binding assays. We conclude that an extra cysteine in the GPIHBP1 Ly6 motif results in multimerization of GPIHBP1, defective LPL binding, and severe hypertriglyceridemia.

Highlights

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) binds lipoprotein lipase (LPL) and transports it to the capillary lumen
Identification of a GPIHBP1 Missense Mutation—Ninetytwo patients with severe hypertriglyceridemia but lacking mutations in LPL, APOC2, or APOA5 were screened for GPIHBP1 mutations
To determine whether multimerization occurs inside the cell or only after reaching the cell surface, we examined the migration pattern under nonreducing conditions of cell lysates prepared from CHO-K1 cells that had been transfected with wildtype GPIHBP1 or GPIHBP1-S107C and treated with Phosphatidylinositol-specific Phospholipase C (PIPLC)

Summary

Background

GPIHBP1 binds lipoprotein lipase (LPL) and transports it to the capillary lumen. Results: A GPIHBP1 missense mutation (S107C) leads to the formation of GPIHBP1 multimers that cannot bind LPL. The ability of GPIHBP1 to bind LPL depends on the Ly6 domain, a threefingered structure containing 10 cysteines and a conserved pattern of disulfide bond formation. Many mammalian Ly6 proteins, including GPIHBP1, have a total of 10 cysteines, leading to the formation of an extra disulfide bond that stabilizes the first loop in the threefingered structural motif. Six GPIHBP1 missense mutations, all involving conserved amino acids in the Ly6 domain, have already been linked to chylomicronemia in humans [11,12,13,14,15,16,17,18] In four of these cases, the mutant GPIHBP1 was tested and shown to lack the ability to bind LPL [12, 13, 15, 16]. Our studies revealed the mechanism by which this GPIHBP1 mutation leads to chylomicronemia

EXPERIMENTAL PROCEDURES

RESULTS

I-2 II-1 II-2 II-6 II-7 II-8 II-9 II-10 III-1 III-2

DISCUSSION