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2.
J Lipid Res ; 50(12): 2421-9, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19542565

ABSTRACT

Glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) binds both LPL and chylomicrons, suggesting that GPIHBP1 is a platform for LPL-dependent processing of triglyceride (TG)-rich lipoproteins. Here, we investigated whether GPIHBP1 affects LPL activity in the absence and presence of LPL inhibitors angiopoietin-like (ANGPTL)3 and ANGPTL4. Like heparin, GPIHBP1 stabilized but did not activate LPL. ANGPTL4 potently inhibited nonstabilized LPL as well as heparin-stabilized LPL but not GPIHBP1-stabilized LPL. Like ANGPTL4, ANGPTL3 inhibited nonstabilized LPL but not GPIHBP1-stabilized LPL. ANGPTL3 also inhibited heparin-stabilized LPL but with less potency than nonstabilized LPL. Consistent with these in vitro findings, fasting serum TGs of Angptl4(-/-)/Gpihbp1(-/-) mice were lower than those of Gpihbp1(-/-) mice and approached those of wild-type littermates. In contrast, serum TGs of Angptl3(-/-)/Gpihbp1(-/-) mice were only slightly lower than those of Gpihbp1(-/-) mice. Treating Gpihbp1(-/-) mice with ANGPTL4- or ANGPTL3-neutralizing antibodies recapitulated the double knockout phenotypes. These data suggest that GPIHBP1 functions as an LPL stabilizer. Moreover, therapeutic agents that prevent LPL inhibition by ANGPTL4 or, to a lesser extent, ANGPTL3, may benefit individuals with hyperlipidemia caused by gene mutations associated with decreased LPL stability.


Subject(s)
Angiopoietins/metabolism , Carrier Proteins/metabolism , Lipoprotein Lipase/metabolism , Receptors, Lipoprotein/metabolism , Angiopoietin-Like Protein 3 , Angiopoietin-Like Protein 4 , Angiopoietin-like Proteins , Angiopoietins/deficiency , Animals , Cattle , Enzyme Stability , Humans , Lipoprotein Lipase/antagonists & inhibitors , Mice , Mice, Knockout , Receptors, Lipoprotein/deficiency , Recombinant Proteins/metabolism
3.
J Biol Chem ; 284(20): 13735-13745, 2009 May 15.
Article in English | MEDLINE | ID: mdl-19318355

ABSTRACT

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln(29)-His(53), which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu(32)-His(55). We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3(-/-) mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.


Subject(s)
Angiopoietins/metabolism , Lipoprotein Lipase/antagonists & inhibitors , Lipoprotein Lipase/metabolism , Angiopoietin-Like Protein 3 , Angiopoietin-Like Protein 4 , Angiopoietin-like Proteins , Angiopoietins/genetics , Angiopoietins/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Humans , Hyperlipidemias/drug therapy , Hyperlipidemias/genetics , Hyperlipidemias/immunology , Hyperlipidemias/metabolism , Lipoprotein Lipase/genetics , Lipoprotein Lipase/immunology , Mice , Mice, Knockout , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Triglycerides/genetics , Triglycerides/immunology , Triglycerides/metabolism
4.
J Am Chem Soc ; 129(51): 16175-82, 2007 Dec 26.
Article in English | MEDLINE | ID: mdl-18044899

ABSTRACT

Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.


Subject(s)
Proteins/metabolism , Catalysis , Models, Molecular , Proteins/chemistry , Substrate Specificity
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