Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2
1998; Elsevier BV; Volume: 39; Issue: 12 Linguagem: Inglês
10.1016/s0022-2275(20)33314-9
ISSN1539-7262
AutoresShau‐Feng Chang, Berthold Reich, John D. Brunzell, Hans Will,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoMonoclonal antibody (MAb) 5D2 recognizes lipoprotein lipases (LPL) from different species but not related lipases. This MAb is a unique reagent, used world-wide, because it differentiates between monomeric inactive and dimeric active LPL, inhibits human LPL enzyme activity, and binds to C-terminal LPL sequences involved in interactions with lipoproteins, lipoprotein receptors, and heparin. In this study we have analyzed the fine specificity of the MAb epitope recognition in order to better understand its functional properties and species-specific LPL immune reactivity. In peptide scan assays, MAb 5D2 reacted with all, except two, 13 amino acid-long peptides located between positions 380 and 410. Peptides from the amino terminal end of this region reacted more strongly than those from the carboxyl terminal end. Furthermore, only a peptide from the amino terminal end competed effectively with the binding of MAb 5D2 to native LPL bound to microtiter plates or nitrocellulose. A systematic peptide mutagenesis study indicated that 8 amino acids of the reactive region, mainly located in the amino terminal end, are critical for binding and probably directly interact with MAb 5D2. The experimentally determined antigenicities of species-specific LPL peptides and of the corresponding denatured full-length LPL proteins on immunoblots were consistent with these findings. According to a proposed 3D-model for LPL, only the amino terminal end of the antigenic region is easily surface-accessible. These data combined with 3D-modelling of monoclonal antibody (MAb)–lipoprotein lipase (LPL) protein interaction provide new insight into the known biological effects of MAb 5D2 on LPL and the antigenic determinants that are recognized.—Chang, S-F., B. Reich, J. D. Brunzell, and H. Will. Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2. J. Lipid Res. 1998. 39: 2350–2359. Monoclonal antibody (MAb) 5D2 recognizes lipoprotein lipases (LPL) from different species but not related lipases. This MAb is a unique reagent, used world-wide, because it differentiates between monomeric inactive and dimeric active LPL, inhibits human LPL enzyme activity, and binds to C-terminal LPL sequences involved in interactions with lipoproteins, lipoprotein receptors, and heparin. In this study we have analyzed the fine specificity of the MAb epitope recognition in order to better understand its functional properties and species-specific LPL immune reactivity. In peptide scan assays, MAb 5D2 reacted with all, except two, 13 amino acid-long peptides located between positions 380 and 410. Peptides from the amino terminal end of this region reacted more strongly than those from the carboxyl terminal end. Furthermore, only a peptide from the amino terminal end competed effectively with the binding of MAb 5D2 to native LPL bound to microtiter plates or nitrocellulose. A systematic peptide mutagenesis study indicated that 8 amino acids of the reactive region, mainly located in the amino terminal end, are critical for binding and probably directly interact with MAb 5D2. The experimentally determined antigenicities of species-specific LPL peptides and of the corresponding denatured full-length LPL proteins on immunoblots were consistent with these findings. According to a proposed 3D-model for LPL, only the amino terminal end of the antigenic region is easily surface-accessible. These data combined with 3D-modelling of monoclonal antibody (MAb)–lipoprotein lipase (LPL) protein interaction provide new insight into the known biological effects of MAb 5D2 on LPL and the antigenic determinants that are recognized. —Chang, S-F., B. Reich, J. D. Brunzell, and H. Will. Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2. J. Lipid Res. 1998. 39: 2350–2359. Lipoprotein lipase (LPL, EC 3.1.1.34) plays a pivotal role in lipoprotein metabolism both as a triglyceride hydrolyzing enzyme and as a mediator of interactions of lipoproteins with cell surfaces and receptors (1Auwerx J. Leroy P. Schoonjans K. Lipoprotein lipase: recent contributions from molecular biology.Crit. Rev. Clin. Lab. Sci. 1992; 29: 243-268Google Scholar, 2Olivecrona G. Olivecrona T. Triglyceride lipases and atherosclerosis.Curr. Opin. Lipidol. 1995; 6: 291-305Google Scholar, 3Santamarina-Fojo S. Dugi K.A. Structure, function and role of lipoprotein lipase in lipoprotein metabolism.Curr. Opin. Lipidol. 1994; 5: 117-125Google Scholar). The fact that human LPL deficiency results in hypertriglyceridemia, a risk factor for atherosclerosis, and the observation that LPL knock-out mice are not viable underscore the importance of this enzyme in physiology (4Coleman T. Seip R.L. Gimble J.M. Lee D. Maeda N. Semenkovich C.F. COOH-terminal disruption of lipoprotein lipase in mice is lethal in homozygotes, but heterozygotes have elevated triglycerides and impaired enzyme activity.J. Biol. Chem. 1995; 270: 12518-12525Google Scholar). The amino acid sequences of the human LPL (hLPL) (5Wion K.L. Kirchgessner T.G. Lusis A.J. Schotz M.C. Lawn R.M. Human lipoprotein lipase complementary DNA sequence.Science. 1987; 235: 1638-1641Google Scholar) and a number of sequence polymorphisms as well as clinically relevant mutations are known (1Auwerx J. Leroy P. Schoonjans K. Lipoprotein lipase: recent contributions from molecular biology.Crit. Rev. Clin. Lab. Sci. 1992; 29: 243-268Google Scholar, 6Hayden M.R. Ma Y. Brunzell J. Henderson H.E. Genetic variants affecting human lipoprotein lipase and hepatic lipase.Curr. Opin. Lipidol. 1991; 2: 104-109Google Scholar, 7Brunzell J.D. Lipoprotein lipase deficiency and other causes of the chylomicronemia syndrome.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill Book Co., New York1995: 1913-1932Google Scholar). In addition, the LPL protein sequences of various animal species such as rat (8Brault D. Noe L. Etienne J. Hamelin J. Raisonnier A. Souli A. Chuat J.C. Dugail I. Quignard-Boulange A. Lavau M. Galibert F. Sequence of rat lipoprotein lipase-encoding cDNA.Gene. 1992; 121: 237-246Google Scholar), bovine (9Senda M. Oka K. Brown W.V. Qasba P.K. Furuichi Y. Molecular cloning and sequence of a cDNA coding for bovine lipoprotein lipase.Proc. Natl. Acad. Sci. USA. 1987; 84: 4369-4373Google Scholar), sheep (10Edwards W.D. Daniels S.W. Page R.A. Volpe C.P. Kille P. Sweeney G.E. Cryer A. Cloning and sequencing of a full length cDNA encoding bovine lipoprotein lipase.Biochim. Biophys. Acta. 1993; 1171: 167-170Google Scholar), pig (11Harbitz I. Karistensen T. Kran S. Davies W. Isolation and sequencing of porcine lipoprotein lipase cDNA and its use in multiallelic restriction fragment length polymorphism detection.Anim. Genet. 1992; 23: 517-522Google Scholar), baboon (12Cole S.A. Hixson J.E. Baboon lipoprotein lipase: cDNA sequence and variable tissue-specific expression of two transcripts.Gene. 1995; 161: 265-269Google Scholar), cat (13Ginzinger D.G. Lewis M.E.S. Ma Y. Jones B.R. Liu G. Jones S.D. Hayden M.R. A mutation in the lipoprotein lipase gene is the molecular basis of chylomicronemia in a colony of domestic cats.J. Clin. Invest. 1996; 97: 1257-1266Google Scholar), mink (14Lindberg A. Nordstoga K. Christophersen B. Savonen R. van Tol A. Olivecrona G. A mutation in the lipoprotein lipase gene associated with hyperlipoproteinemia type I in mink: studies on lipid and lipase levels in heterozygotes.Int. J. Mol. Med. 1998; 1: 529-538Google Scholar), mouse (15Kirchgessner T.G. Svenson K.L. Lusis A.L. Schotz M.C. The sequence of cDNA encoding lipoprotein lipase: a member of a lipase gene family.J. Biol. Chem. 1987; 262: 8463-8466Google Scholar), guinea pig (16Enerbaeck S. Semb H. Bengtsson-Olivecrona G. Carlsson P. Hermansson M.L. Olivecrona T. Bjursell G. Molecular cloning and sequence analysis of cDNA encoding lipoprotein lipase of guinea pig.Gene. 1987; 58: 1-12Google Scholar), and chicken (17Cooper D.A. Lu S.C. Viswanath R. Freiman R.N. Bensadoun A. The structure and complete nucleotide sequence of the avian lipoprotein lipase gene.Biochim. Biophys. Acta. 1992; 1129: 166-171Google Scholar) have been published. Similar to the situation for human LPL (2Olivecrona G. Olivecrona T. Triglyceride lipases and atherosclerosis.Curr. Opin. Lipidol. 1995; 6: 291-305Google Scholar, 6Hayden M.R. Ma Y. Brunzell J. Henderson H.E. Genetic variants affecting human lipoprotein lipase and hepatic lipase.Curr. Opin. Lipidol. 1991; 2: 104-109Google Scholar, 7Brunzell J.D. Lipoprotein lipase deficiency and other causes of the chylomicronemia syndrome.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill Book Co., New York1995: 1913-1932Google Scholar), naturally occurring mutations in LPL, which result in hypertriglyceridemia, have also been reported for cat (13Ginzinger D.G. Lewis M.E.S. Ma Y. Jones B.R. Liu G. Jones S.D. Hayden M.R. A mutation in the lipoprotein lipase gene is the molecular basis of chylomicronemia in a colony of domestic cats.J. Clin. Invest. 1996; 97: 1257-1266Google Scholar) and mink (18Christophersen B. Nordstoga K. Shen Y. Olivecrona T. Olivecrona G. Lipoprotein lipase deficiency with pancreatitis in mink: biochemical characterization and pathology.J. Lipid Res. 1997; 38: 837-846Google Scholar). The observation of strong sequence similarities between the coding region and gene organization of LPL, hepatic triglyceride lipase (HL, EC 3.1.1.32), and pancreatic lipase (PL, EC 3.1.1.3) indicates that all three enzymes are members of a gene family (19Ameis D. Stahnke G. Kobayashi J. McLean J. Lee G. Büscher M. Schotz M.C. Will H. Isolation and characterization of the human hepatic lipase gene.J. Biol. Chem. 1990; 265: 6552-6555Google Scholar, 20Datta S. Luo C.C. Li H-H. van Tuinen P. Ledbetter D.H. Brown M.A. Chen S-H. Liu S-W. Chan L. Human hepatic lipase. Cloned cDNA sequence, restriction fragment length polymorphisms, chromosomal localization, and evolutionary relationships with lipoprotein lipase and pancreatic lipase.J. Biol. Chem. 1988; 263: 1107-1110Google Scholar, 21Hide W.A. Chan L. Li W.H. Structure and evolution of the lipase superfamily.J. Lipid Res. 1992; 33: 167-178Google Scholar, 22Stahnke G. Sprengel R. Augustin J. Will H. Human hepatic triglyceride lipase: cDNA cloning amino acid sequence and expression in a cultured cell line.Differentiation. 1987; 35: 45-52Google Scholar). Based on this information and on the known three dimensional (3-D) structure of PL, a 3D-structure similar to PL was proposed for LPL (23van Tilbeurgh H. Roussel A. Lalouel J-M. Cambillau C. Lipoprotein lipase. Molecular model based on the pancreatic lipase X-ray structure: consequences for heparin binding and catalysis.J. Biol. Chem. 1994; 269: 4626-4633Google Scholar) consisting of N- and C-terminal folding domains. Enzymatically active LPL appears to be a non-covalently linked homodimer (24Osborne J.C.J. Bengtsson-Olivecrona G. Lee N.S. Olivecrona T. Studies on inactivation of lipoprotein lipase: role of the dimer to monomer dissociation.Biochemistry. 1985; 24: 5606-5611Google Scholar, 25Iverius P-H. Ostlund-Lindqvist A-M. Lipoprotein lipase from bovine milk: isolation procedure, chemical characterization, and molecular weight analysis.J. Biol. Chem. 1976; 251: 7791-7795Google Scholar, 26Babirak S.P. Iverius P-H. Fujimoto W.Y. Brunzell J.D. Detection and characterization of the heterozygote state for lipoprotein lipase deficiency.Arteriosclerosis. 1989; 9: 326-334Google Scholar) with a head-to-tail subunit orientation (27Wong H. Yang D. Hill J.S. Davis R.C. Nikazy J. Schotz M.C. A molecular biology-based approach to resolve the subunit orientation of lipoprotein lipase.Proc. Natl. Acad. Sci. USA. 1997; 94: 5594-5598Google Scholar) that rapidly dissociates into inactive monomers (24Osborne J.C.J. Bengtsson-Olivecrona G. Lee N.S. Olivecrona T. Studies on inactivation of lipoprotein lipase: role of the dimer to monomer dissociation.Biochemistry. 1985; 24: 5606-5611Google Scholar, 28Peterson J. Fujimoto W.Y. Brunzell J.D. Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies.J. Lipid Res. 1992; 33: 1165-1170Google Scholar). However, evidence for enzymatically active monomeric human LPL has also been presented (29Ikeda Y. Takagi A. Yamamoto A. Purification and characterization of lipoprotein and hepatic triglyceride lipase from human postheparin plasma: production of monospecific antibody to the individual lipase.Biochim. Biophys. Acta. 1989; 1003: 254-269Google Scholar). A strong argument for the dimer model is the immune reactivity of native human LPL in a sandwich ELISA with MAb 5D2 (28Peterson J. Fujimoto W.Y. Brunzell J.D. Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies.J. Lipid Res. 1992; 33: 1165-1170Google Scholar), provided this MAb recognizes only a single epitope in each monomer of the native dimeric LPL enzyme. As the reactivity of only two short peptide sequences of LPL have been tested to date (30Liu M-S. Ma Y. Hayden M.R. Brunzell J.D. Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity.Biochim. Biophys. Acta. 1992; 1128: 113-115Google Scholar), the existence of additional epitopes recognized by MAb 5D2 is not excluded. The model of the 3-D structure for LPL has been very useful in the designing of experiments and understanding the data obtained from functional analysis studies performed with naturally occurring and artificially created LPL mutants (31Previato L. Guardamagna O. Dugi K.A. Ronan R. Talley G.D. Santamarina-Fojo S. Brewer Jr., H.B. A novel missense mutation in the C-terminal domain of lipoprotein lipase (Glu410 → Val) leads to enzyme inactivation and familial chylomicronemia.J. Lipid Res. 1994; 35: 1552-1560Google Scholar, 32Lookene A. Groot N.B. Kastelein J.J.P. Olivecrona G. Bruin T. Mutation of tryptophan residues in lipoprotein lipase.J. Biol. Chem. 1997; 272: 766-772Google Scholar, 33Wong H. Davis R.C. Thuren T. Goers J.W. Nikazy J. Waite M. Schotz M.C. Lipoprotein lipase domain function.J. Biol. Chem. 1994; 269: 10319-10323Google Scholar, 34Krapp A. Zhang H. Ginzinger D. Liu M-S. Lindberg A. Olivecrona G. Hayden M.R. Beisiegel U. Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells.J. Lipid Res. 1995; 36: 2362-2373Google Scholar, 35Kazaki K. Gotoda T. Kawamura M. Shimano H. Yazaki Y. Ouchi Y. Orimo H. Yamada N. Mutational analysis of human lipoprotein lipase by carboxy-terminal truncation.J. Lipid Res. 1993; 34: 1765-1772Google Scholar). Accordingly, the N-terminal domain (aa 1–312) contains the catalytic center with a covering loop important for interaction with lipid substrates, heparin binding sites, and a binding site for the cofactor apolipoprotein C-II (3Santamarina-Fojo S. Dugi K.A. Structure, function and role of lipoprotein lipase in lipoprotein metabolism.Curr. Opin. Lipidol. 1994; 5: 117-125Google Scholar). The C-terminal domain (aa 313–448) was shown to contain binding sites for lipoproteins, for the α2-macroglobulin receptor/low density lipoprotein receptor-related protein, and possibly for heparin (36Nielsen M.S. Brejning J. García R. Zhang H. Hayden M.R. Vilaró S. Gliemann J. Segments in the C-terminal folding domain of lipoprotein lipase important for binding to the low density lipoprotein receptor-related protein and to heparin sulfate proteoglycans.J. Biol. Chem. 1997; 272: 5821-5827Google Scholar, 37Williams S.E. Inoue I. Tran H. Fry G.L. Pladet M.W. Iverius P-H. Lalouel J-M. Chappell D.A. Strickland D.K. The carboxyl-terminal domain of lipoprotein lipase binds to the low density lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP) and mediates binding of normal very low density lipoproteins to LRP.J. Biol. Chem. 1994; 269: 8653-8658Google Scholar, 38Medh J.D. Bowen S.L. Ruben G.L. Fry S. Andracki M. Inoue I. Lalouel J-M. Strickland D.K. Chappell D.A. Lipoprotein lipase binds to low density lipoprotein receptors and induces receptor-mediated catabolism of very low density liporoteins in vitro.J. Biol. Chem. 1996; 271: 17073-17080Google Scholar). As the sequence similarity between the C-terminal sequences of LPL and PL is much weaker than that between the N-terminal sequences, the folding predictions concerning the C-terminal LPL domain are less certain. In agreement with the proposed folding of the C-terminal LPL domain is the observation that chymotrypsin cleaves human LPL between positions 390/391 and 391/392. In the 3-D model both cleavage sites are located in a surface exposed loop (23van Tilbeurgh H. Roussel A. Lalouel J-M. Cambillau C. Lipoprotein lipase. Molecular model based on the pancreatic lipase X-ray structure: consequences for heparin binding and catalysis.J. Biol. Chem. 1994; 269: 4626-4633Google Scholar, 32Lookene A. Groot N.B. Kastelein J.J.P. Olivecrona G. Bruin T. Mutation of tryptophan residues in lipoprotein lipase.J. Biol. Chem. 1997; 272: 766-772Google Scholar). In contrast, the binding site of the LPL-specific MAb 5D2 previously roughly mapped to aa 397–407 at the C-terminal domain (30Liu M-S. Ma Y. Hayden M.R. Brunzell J.D. Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity.Biochim. Biophys. Acta. 1992; 1128: 113-115Google Scholar) is not located at the surface (see below). Therefore, we reasoned that either the proposed 3-D structure does not fully reflect the structure of the native enzyme or the epitope mapped for MAb 5D2 is only accessible in partially denatured LPL. Based on the observation of reduced immune reactivity of LPL mutated in amino acids 390, 394, and 393/394 it has recently been speculated that the epitope recognized by MAb 5D2 may be longer than reported (32Lookene A. Groot N.B. Kastelein J.J.P. Olivecrona G. Bruin T. Mutation of tryptophan residues in lipoprotein lipase.J. Biol. Chem. 1997; 272: 766-772Google Scholar). Alternatively, the same authors suggested that these mutations, which are located close to the epitope mapped previously (30Liu M-S. Ma Y. Hayden M.R. Brunzell J.D. Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity.Biochim. Biophys. Acta. 1992; 1128: 113-115Google Scholar), may change the conformation of the epitope and thus abrogate binding of the MAb 5D2 (32Lookene A. Groot N.B. Kastelein J.J.P. Olivecrona G. Bruin T. Mutation of tryptophan residues in lipoprotein lipase.J. Biol. Chem. 1997; 272: 766-772Google Scholar). The LPL-specific MAb 5D2 has several unique features and is one of the most frequently used immunological reagents in LPL research world-wide. Although this MAb was originally produced against bovine LPL, it crossreacts strongly with human LPL and with LPL from all animals (except mice) tested so far (18Christophersen B. Nordstoga K. Shen Y. Olivecrona T. Olivecrona G. Lipoprotein lipase deficiency with pancreatitis in mink: biochemical characterization and pathology.J. Lipid Res. 1997; 38: 837-846Google Scholar, 30Liu M-S. Ma Y. Hayden M.R. Brunzell J.D. Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity.Biochim. Biophys. Acta. 1992; 1128: 113-115Google Scholar). No immunological crossreaction has ever been observed with related hepatic lipases or pancreatic lipase. MAb 5D2 was reported to recognize the enzymatically active LPL dimer in a sandwich enzyme-linked assay (ELISA) whereas the inactive monomer is recognized in a sandwich ELISA only when a second LPL-specific antibody is also used (28Peterson J. Fujimoto W.Y. Brunzell J.D. Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies.J. Lipid Res. 1992; 33: 1165-1170Google Scholar). Therefore, this MAb is often used to determine the ratio of enzymatically active and inactive LPL as usually present simultaneously in sera and supernatants of transfected cells (32Lookene A. Groot N.B. Kastelein J.J.P. Olivecrona G. Bruin T. Mutation of tryptophan residues in lipoprotein lipase.J. Biol. Chem. 1997; 272: 766-772Google Scholar, 34Krapp A. Zhang H. Ginzinger D. Liu M-S. Lindberg A. Olivecrona G. Hayden M.R. Beisiegel U. Structural features in lipoprotein lipase necessary for the mediation of lipoprotein uptake into cells.J. Lipid Res. 1995; 36: 2362-2373Google Scholar). Binding of MAb 5D2 to native LPL is known to inhibit its hydrolytic activity towards long chain triglycerides (triolein as substrate) but not its esterase activity towards short chain substrates such as tributyrin (33Wong H. Davis R.C. Thuren T. Goers J.W. Nikazy J. Waite M. Schotz M.C. Lipoprotein lipase domain function.J. Biol. Chem. 1994; 269: 10319-10323Google Scholar). The inhibition is not due to dissociation of the LPL dimers but presumably due to interference with the function of the second lipid binding site located at the C-terminal domain (33Wong H. Davis R.C. Thuren T. Goers J.W. Nikazy J. Waite M. Schotz M.C. Lipoprotein lipase domain function.J. Biol. Chem. 1994; 269: 10319-10323Google Scholar). MAb 5D2 is also often one of the decisive reagents for the determination of the mass of LPL in sandwich ELISAs based on two MAbs including MAb 5D2 (28Peterson J. Fujimoto W.Y. Brunzell J.D. Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies.J. Lipid Res. 1992; 33: 1165-1170Google Scholar). This is possible because MAb 5D2 does not only bind to native LPL dimer but also to heat- or GuHCl-denatured LPL (28Peterson J. Fujimoto W.Y. Brunzell J.D. Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies.J. Lipid Res. 1992; 33: 1165-1170Google Scholar). Moreover, MAb 5D2 also recognizes nitrocellulose-fixed LPL on immunoblots although the enzyme is boiled and denatured with sodium dodecylsulfate before blotting (28Peterson J. Fujimoto W.Y. Brunzell J.D. Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies.J. Lipid Res. 1992; 33: 1165-1170Google Scholar). Taken together, this information clearly indicates that MAb 5D2 has unique binding characteristics. The many known and interesting biological features of MAb 5D2 combined with the wealth of information about the functional domains of LPL as well as the availability of a 3-D model for LPL motivated us to re-investigate and map in detail the epitope recognized by the LPL-specific MAb 5D2. The mapping data obtained indicate that the LPL specific sequences bound by MAb 5D2 are more complex than previously elucidated. Moreover, they provide explanations for the known functions of MAb 5D2 and are consistent with 3-D structure predictions for LPL. In total, 232 peptides, each 13 amino acids long with an overlap of 11 amino acids to the next peptide and spanning the whole human LPL protein sequence, were synthesized on cellulose filters using previously reported methods (39Kramer A. Vakalopoulou E. Schleuning W-D. Schneider-Mergener J. A general route to fingerprint analyses of peptide–antibody interactions using a clustered amino acid peptide library: comparison with a phage display library.Mol. Immunol. 1995; 32: 459-465Google Scholar, 40Frank R. Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support.Tetrahedron. 1992; 48: 9217-9232Google Scholar, 41Derewenda Z.S. Cambillau C. Effects of gene mutations in lipoprotein and hepatic lipases as interpreted by a molecular model of the pancreatic triglyceride lipase.J. Biol. Chem. 1991; 266: 23112-23119Google Scholar) (Jerini Biotools Co., Berlin, Germany). The peptides were designated with P followed by a number indicating the first amino acid of the full length human LPL protein (position one is the first amino acid of the mature protein and position −27 the first amino acid of the signal sequence). The cellulose filters containing the hLPL peptides were incubated with the monoclonal 5D2 antibody (MAb 5D2) (1 μg/ml) in TBS buffer (10 mm Tris-HCl, 150 mm NaCl, pH 7.6). Binding was detected using alkaline phophatase-labeled or horseradish peroxidase (HRP)-labeled anti-mouse antibodies (Dianova, Hamburg, Germany). Keyhole limpet haemocyanin (KLH)-coupled peptides P384/395 (SDSYFSWSDWWS) and P396/410 (SPGFAIQKIRVKAGE) were dissolved in PBS containing 1% SDS. Microtiter plates (Nunc, Denmark) were coated with the amount of peptides indicated and incubated at 37°C for 2 h. After washing once with EPBS (10 mm NaPO4, 2 mm KH2PO4, 165 mm NaCl, 3.2 m KCl, pH 7.4) containing 0.1% Tween 20, the plates were blocked overnight with EPBS/2% BSA at 4°C. HRP-labeled anti-LPL MAb 5D2 (70 pg/ml in EPBS/0.1% Tween 20) was then added to the microtiter wells. The plates were then incubated at room temperature for 4 h and washed 5 times with EPBS/0.1% Tween 20. HRP enzyme activity was quantitated using as a substrate 0.5 mg/ml o-phenylenediamine in 0.01% H2O2, 0.1 m citrate–phosphate buffer, pH 5.0, incubated for 10 min. Absorbance was measured at 490 nm. Plates were coated with partially purified LPL (kindly provided by U. Beisiegel, Hamburg, Germany, G. Olivecrona, Umeå, Sweden, and H. Jansen, Rotterdam, Holland) diluted in EPBS (50 ng/well) at 8°C overnight. After washing with EPBS, the plate was blocked with EPBS/2% BSA overnight. HRP-labeled MAb 5D2 preincubated for 1 h at room temperature with different concentrations of the peptides were then added to the microtiter wells and incubated for 3 h at room temperature. The plates were finally washed and the HRP enzyme activity was measured as described above. The plates were coated with 50 μl/well of MAb 5D2 (4 μg/ml) overnight at 8°C, and then blocked with EPBS/2% BSA. Partially purified LPL (50 ng/well diluted in EPBS) was then added and the plates were incubated overnight at 8°C. After washing, HRP-labeled MAb 5D2 preincubated with peptides as described above was added to the microtiter plates. After incubation of the plates at room temperature for 3 h, the plates were washed again as described above, and the HRP enzyme activity was determined as described above. The proteins were either separated by SDS-PAGE and then transferred onto nitrocellulose membrane, or alternatively, the proteins were directly spotted onto the membranes. Non-saturated protein binding sites on the membranes were then blocked with 5% milk powder in H2O at room temperature for 2 h, and finally incubated for 2 h at room temperature with MAb 5D2 (1 μg/ml in TBS containing 1% milk). After washing the membranes thrice with TBS/0.1% Tween for 10 min and thrice with TBS for 10 min, they were incubated at room temperature for 2 h with HRP-labeled anti-mouse Ab (diluted 1: 50,000 in TBS/1% milk). The membranes were then washed with TBS/0.1% Tween and TBS as described above. Bound antibodies were detected by chemiluminescence using a commercially available kit (ECL-kit, Amersham Buchler, Braunschweig, Germany) and exposure to X-ray films. The 3D-model of lipases of hLPL published previously (23van Tilbeurgh H. Roussel A. Lalouel J-M. Cambillau C. Lipoprotein lipase. Molecular model based on the pancreatic lipase X-ray structure: consequences for heparin binding and catalysis.J. Biol. Chem. 1994; 269: 4626-4633Google Scholar) was drawn using the molecular modeling program SYBYL on a Silicon Graphics INDY workstation. Functional regions and amino acids outlined in color are taken from published data (3Santamarina-Fojo S. Dugi K.A. Structure, function and role of lipoprotein lipase in lipoprotein metabolism.Curr. Opin. Lipidol. 1994; 5: 117-125Google Scholar, 6Hayden M.R. Ma Y. Brunzell J. Henderson H.E. Genetic variants affecting human lipoprotein lipase and hepatic lipase.Curr. Opin. Lipidol. 1991; 2: 104-109Google Scholar, 23van Tilbeurgh H. Roussel A. Lalouel J-M. Cambillau C. Lipoprotein lipase. Molecular model based on the pancreatic lipase X-ray structure: consequences for heparin binding and catalysis.J. Biol. Chem. 1994; 269: 4626-4633Google Scholar, 31Previato L. Guardamagna O. Dugi K.A. Ronan R. Talley G.D. Santamarina-Fojo S. Brewer Jr., H.B. A novel missense mutation in the C-terminal domain of lipoprotein lipase (Glu410 → Val) leads to enzyme inactivation and familial chylomicronemia.J. Lipid Res. 1994; 35: 1552-1560Google Scholar). The 3D-structure of the Fab fragment of MAb 59.1 (42Ghiara J.B. Stura E.A. Stanfield R.L. Profy A.T. Wilson I.A. Crystal structure of the principal neutralization site of HIV-1.Science. 1994; 264: 82-85Google Scholar) available in the EMBL data bank and known to bind a short peptide (42Ghiara J.B. Stura E.A. Stanfield R.L. Profy A.T. Wilson I.A. Crystal structure of the principal neutralization site of HIV-1.Science. 1994; 264: 82-85Google Scholar) was used to outline the likely steric orientation and size dimensions of the hLPL/MAb 5D2 complex. Similarly, the proposed structure of a dodecamer heparin molecule (43Mulloy B. Forster M.J. Jones C. Davies D.B. N. m. r. and molecular-modelling studies of the solution conformation of heparin.Biochem. J. 1993; 293: 849-858Google Scholar) has been aligned rather arbitrarily along the backbone of hLPL in close proximity to the known heparin-binding sequences (23van Tilbeurgh H. Roussel A. Lalouel J-M. Cambillau C. Lipoprotein lipase. Molecular model based on the pancreatic lipase X-ray structure: consequences for heparin binding and catalysis.J. Biol. Chem. 1994; 269: 4626-4633Google Scholar). In order to search systematically for sequences on human LPL that a
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