Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2

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. 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. 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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), mutations in LPL, in hypertriglyceridemia, have for Ma Y. G. M.R. A in the lipoprotein lipase is the molecular of chylomicronemia in a of Clin. Scholar) and B. K. Y. Olivecrona T. Olivecrona G. Lipoprotein lipase deficiency with in characterization and Lipid Res. Scholar). The observation of sequence between the coding region and of LPL, hepatic triglyceride lipase EC and lipase EC that are of a D. G. J. J. Lee G. M. Schotz M.C. H. Isolation and characterization of the human hepatic lipase Biol. Chem. Scholar, S. P. Brown L. Human hepatic cDNA restriction fragment length and with lipoprotein lipase and Biol. 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A. and characterization of lipoprotein and hepatic triglyceride lipase from human of antibody to the lipase.Biochim. Biophys. Acta. Scholar). A for the is the immune of native human LPL in a with MAb 5D2 J. Brunzell J.D. Human lipoprotein lipase: of activity, and as with monoclonal Lipid Res. 1992; Scholar), this MAb recognizes only a epitope in of the native dimeric LPL the of only peptide sequences of LPL have to Ma Y. M.R. Brunzell J.D. of the epitope on lipoprotein lipase a monoclonal antibody inhibits lipase Biophys. Acta. 1992; Scholar), the of MAb 5D2 is not The of the for LPL in the of and the data from functional with and LPL L. Dugi K.A. S. A in the C-terminal of lipoprotein lipase to enzyme and Lipid Res. 1994; Scholar, A. Olivecrona G. T. of in lipoprotein Biol. Chem. Scholar, H. T. J. M. Schotz M.C. Lipoprotein lipase Biol. Chem. 1994; Scholar, A. H. D. A. Olivecrona G. M.R. in lipoprotein lipase for the of lipoprotein into Lipid Res. 1995; Scholar, K. T. M. H. Y. Y. H. 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Chem. 1994; Scholar, A. Olivecrona G. T. of in lipoprotein Biol. Chem. Scholar). In the binding site of the MAb 5D2 to the C-terminal Ma Y. M.R. Brunzell J.D. of the epitope on lipoprotein lipase a monoclonal antibody inhibits lipase Biophys. Acta. 1992; Scholar) is not located the we that the proposed not the of the native enzyme or the epitope for MAb 5D2 is only in denatured on the observation of immune of LPL in amino acids and it that the epitope MAb 5D2 than A. Olivecrona G. T. of in lipoprotein Biol. Chem. Scholar). the that these are located to the epitope Ma Y. M.R. Brunzell J.D. of the epitope on lipoprotein lipase a monoclonal antibody inhibits lipase Biophys. Acta. 1992; Scholar), the of the epitope and binding of the MAb 5D2 A. Olivecrona G. T. of in lipoprotein Biol. Chem. Scholar). The MAb 5D2 unique and is of the used in LPL this MAb bovine LPL, it strongly with human LPL and with LPL from B. K. Y. Olivecrona T. Olivecrona G. Lipoprotein lipase deficiency with in characterization and Lipid Res. Scholar, Ma Y. M.R. Brunzell J.D. of the epitope on lipoprotein lipase a monoclonal antibody inhibits lipase Biophys. Acta. 1992; Scholar). with related hepatic lipases or MAb 5D2 to the active LPL in a the inactive is in a only a antibody is used J. Brunzell J.D. Human lipoprotein lipase: of activity, and as with monoclonal Lipid Res. 1992; Scholar). this MAb is used to the of active and inactive LPL as in and of A. Olivecrona G. T. of in lipoprotein Biol. Chem. Scholar, A. H. D. A. Olivecrona G. M.R. in lipoprotein lipase for the of lipoprotein into Lipid Res. 1995; Scholar). of MAb 5D2 to native LPL is known to its triglycerides as but not its such as H. T. J. M. Schotz M.C. Lipoprotein lipase Biol. Chem. 1994; Scholar). The is not to of the LPL but to with the function of the binding site located the C-terminal H. T. J. M. Schotz M.C. Lipoprotein lipase Biol. Chem. 1994; Scholar). MAb 5D2 is of the for the of the of LPL in on MAb 5D2 J. Brunzell J.D. Human lipoprotein lipase: of activity, and as with monoclonal Lipid Res. 1992; Scholar). This is because MAb 5D2 not only to native LPL but to or LPL J. Brunzell J.D. Human lipoprotein lipase: of activity, and as with monoclonal Lipid Res. 1992; Scholar). MAb 5D2 recognizes LPL on immunoblots the enzyme is and denatured with J. Brunzell J.D. Human lipoprotein lipase: of activity, and as with monoclonal Lipid Res. 1992; Scholar). this that MAb 5D2 unique binding The known and biological of MAb 5D2 combined with the of the functional of LPL as well as the of a for LPL to and in the epitope the MAb 5D2. The data that the LPL sequences bound MAb 5D2 are more than provide for the known of MAb 5D2 and are consistent with for In 13 amino acids with of amino acids to the peptide and the human LPL protein were on A. J. A to of interactions a amino acid peptide with a 1995; Scholar, for the on a 1992; Scholar, of mutations in lipoprotein and hepatic lipases as a molecular of the triglyceride Biol. Chem. 1991; Scholar) Co., The peptides were with a number the amino acid of the full length human LPL protein is the amino acid of the protein and the amino acid of the The the peptides were with the monoclonal 5D2 antibody in or peptides and were in plates were with the of peptides indicated and for with the plates were with MAb 5D2 in to the microtiter The plates were for and with enzyme as a in for were with LPL G. and H. in with the with MAb 5D2 for with different of the peptides were to the microtiter and for The plates were and the enzyme as The plates were with of MAb 5D2 and with LPL in and the plates were MAb 5D2 with peptides as to the microtiter of the plates for the plates were as and the enzyme determined as The proteins were and or the proteins were directly the protein binding on the were with in for and for with MAb 5D2 in the with for and with for were for with in The were with and as were a and to The 3D-model of lipases of H. A. Lipoprotein Molecular on the lipase for binding and Biol. Chem. 1994; Scholar) the molecular on a and amino acids in are from data 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, H. A. Lipoprotein Molecular on the lipase for binding and Biol. Chem. 1994; Scholar, L. Dugi K.A. S. A in the C-terminal of lipoprotein lipase to enzyme and Lipid Res. 1994; Scholar). The of the fragment of MAb R.L. of the site of 1994; Scholar) in the data and known to a peptide R.L. of the site of 1994; Scholar) used to the and of the 5D2 the proposed of a B. Davies N. and of the of J. 1993; Scholar) the of in to the known sequences H. A. Lipoprotein Molecular on the lipase for binding and Biol. Chem. 1994; Scholar). In order to for sequences on human LPL that are MAb peptides the 13 amino acids and with the amino were on a and MAb 5D2 with the and its binding with a on immune of MAb 5D2 with peptides to and in sequence and amino acids 380 to of the LPL protein and The immune for peptide for peptides and and for peptides and These data that MAb 5D2 binds strongly to amino acid sequence to and that amino acids to are to immune reactive are for its we to binding to LPL sequences amino acid to as the epitope MAb 5D2 Ma Y. M.R. Brunzell J.D. of the epitope on lipoprotein lipase a monoclonal antibody inhibits lipase Biophys. Acta. 1992; Scholar), we the of with the corresponding peptides to of we the of the a with MAb 5D2 of and a with of the strongly with peptides to as but in immune with peptide the epitope sequence Ma Y. M.R. Brunzell J.D. of the epitope on lipoprotein lipase a monoclonal antibody inhibits lipase Biophys. Acta. 1992; Scholar). were with peptides and are to both immune reactive as well as with various peptides of these of the peptides of the immune reactive region not in a not it these data that MAb 5D2 binds to LPL peptide sequences amino acids 380 to and to a peptide amino acids to both as with the data that MAb 5D2 to protein The of both epitope sequences as well as the immune of peptides and located the region that MAb 5D2 recognizes both of a LPL epitope from amino acid 380 to 410. the binding of MAb 5D2 to both of the antigenic region in peptides and to the and amino acid of the human were to and used as in In this binding of MAb 5D2 to both peptides of peptide to to the of enzyme binding used as in a the specificity of the binding of peptides and These data that MAb 5D2 binds to both sequences and to the located epitope more strongly than to the epitope located in the C-terminal This is consistent with the data with the binding a we in a peptides and binding of MAb 5D2 to LPL microtiter plates as well as the binding of MAb 5D2 to LPL MAb 5D2 in microtiter In both a with peptide peptide to LPL directly to microtiter plates to and of MAb 5D2 binding with peptide in both to a peptide and we that peptide in with binding of MAb 5D2 to native LPL with this a with LPL results These data that the located epitope the peptide not a in with binding MAb 5D2. In order to provide for the of the data and to the of amino acids for and with binding of MAb 5D2 to human LPL, amino acid of peptides and of peptide in sequence to These peptides and the corresponding were for immune a peptide scan as for peptides the full-length on the a peptide with or a as The with peptide that amino acids positions to and are critical for binding of MAb 5D2 as not amino acid other than to to immune acids and positions and are for binding but other amino acids In amino acids in positions to and of peptide other amino acid the these data that of amino acids and of the LPL protein interact directly with amino acid in the binding of MAb 5D2. Furthermore, as only amino acids or were with binding and both amino acids are a in interaction of the binding site of MAb with the of both amino acids is of amino acids and in positions with binding interaction of the of the of both amino acids with in the binding site of MAb 5D2. The of the of the peptide in from that of as the for the antibody and amino acid positions that in with MAb 5D2 binding were located the C-terminal end acids to as for peptide amino acids positions and of peptide not other amino acid binding of MAb 5D2 acid is critical for binding as it only the amino acid acids positions and other amino acids these data interaction of amino acids and with the binding site of MAb 5D2 and a role for other amino those located the amino terminal end between amino acid positions to The data from the mutagenesis study and the protein sequences for LPL from different species or of LPL from different and of the corresponding LPL peptides corresponding to the amino terminal epitope from human and have a of with MAb 5D2. In those from rat and LPL and not because amino acids and positions and are not with binding as in mutagenesis The of the sequence is the because a peptide with mutations as in to sequence not the of the rat peptide or of the full-length protein to determined experimentally According to data of the mutagenesis recognition of C-terminal epitope peptides from LPL proteins from different species more than that of the amino terminal end because of the LPL proteins have mutations in or more amino acids with binding of MAb 5D2 and The amino acid in in not other amino acid in mutagenesis study immune of the corresponding is in LPL from and The and from the mutagenesis study for the of the LPL peptides from different species corresponding to peptides and of human LPL were experimentally immune reactivity. were on with MAb and with In these MAb 5D2 LPL peptides from and but not from and for the this is consistent with the from the mutagenesis the peptide from those mutagenesis of amino the of both mutations probably a with MAb 5D2 bovine LPL peptides corresponding to of human LPL were used for the from the other only amino acids the amino and the peptide reacted more strongly than the This that binding of MAb 5D2 is strongly on sequence and of the corresponding In the with peptides from the LPL sequences of were with with the rat and with that of the This is consistent with the from the mutagenesis the rat peptide reacted only in this and amino acid and to The of of a corresponding peptide with only in but the of of the peptides with both mutations that a in the peptide for MAb 5D2 binding is interaction of both both the sequence the as well as the of peptides to or not MAb 5D2 we the immune of full-length LPL from and this not experimentally with MAb 5D2 were from of the corresponding and in LPL proteins reacted strongly and with MAb 5D2. These that of the amino terminal end of the antigenic region in these LPL proteins is for immune in or that the of LPL more than epitope for binding MAb 5D2. not and in Ma Y. M.R. Brunzell J.D. of the epitope on lipoprotein lipase a monoclonal antibody inhibits lipase Biophys. Acta. 1992; Scholar), with from of and the of the with MAb 5D2 and the of of the In this study we have that the MAb 5D2 recognizes end of a epitope located between amino acids 380 and 410. amino acids were to for that directly interact with the binding of MAb 5D2. The binding for the amino terminal end of the epitope in the proposed for the of In of the carboxyl terminal end of the to the epitope is not in the of LPL it only in or denatured data and the sequence of the MAb 5D2 epitope in LPL from different were to consistent with its species immune in the with new data as well as with the known of immune of the the data that the epitope MAb 5D2 is more than have for the of the biological properties of MAb 5D2 in to and function of In a the epitope for MAb 5D2 between amino acids and of LPL Ma Y. M.R. Brunzell J.D. of the epitope on lipoprotein lipase a monoclonal antibody inhibits lipase Biophys. Acta. 1992; Scholar). These data were on the of a reactive and a on the of sequence and of LPL from as well as known the of of LPL and human hepatic lipase data are consistent with this but that this region is only of a bound only MAb and probably only in or denatured LPL In to a in the that is critical for immune and into Ma Y. M.R. Brunzell J.D. of the epitope on lipoprotein lipase a monoclonal antibody inhibits lipase Biophys. Acta. 1992; Scholar), we have mutagenesis that of into or other amino acids is with the binding of MAb 5D2. These data and the immune of the corresponding and both have a that is not as critical as and is not the for of immune of of that it is the of to in of the LPL binding MAb 5D2. this is other known LPL is a known and results in of the region of the epitope in as determined not and the mutagenesis study indicated that of other amino acid binding of MAb probably because the of is directly with the binding of MAb 5D2. data are consistent with the of immune of MAb 5D2 with human hepatic lipase and In lipases is sequence with the LPL antigenic region not The reactive of the epitope as for MAb 5D2 to the end of the C-terminal of LPL In the immune of the full-length and LPL proteins in as well as on immunoblots is on these sequences as the reactive region to in LPL of other species is peptide The immune reactive positions and were to to binding of LPL to and to the of the enzyme the A. Olivecrona G. T. of in lipoprotein Biol. Chem. Scholar). The of the as of the binding site of MAb 5D2 strongly that these as well as the triglyceride are directly binding of MAb 5D2 to the located binding The of the of and the proposed of the epitope of MAb 5D2 in the of the of LPL for of the 5D2 we for the dimeric LPL on of H. A. Lipoprotein Molecular on the lipase for binding and Biol. Chem. 1994; Scholar) and recent for the to of the in the dimeric LPL H. D. J. Schotz M.C. A molecular to the of lipoprotein lipase.Proc. Natl. Acad. Sci. USA. Scholar), we the of a 5D2 in the are to such that the active and the binding of both are In we the known of the fragment of a MAb with known and peptide epitope specificity R.L. of the site of 1994; Scholar), as for MAb to the of the epitope in the native LPL MAb 5D2 This of the of the monomeric for the known biological properties of MAb 5D2 and the corresponding on of the C-terminal of MAb 5D2 well in a J. Brunzell J.D. Human lipoprotein lipase: of activity, and as with monoclonal Lipid Res. 1992; Scholar) because the microtiter MAb 5D2 binds to the antigenic of of the LPL the C-terminal antigenic of the other of the LPL on the it for binding of the MAb 5D2 used for binding of MAb 5D2 not with the binding of LPL H. T. J. M. Schotz M.C. Lipoprotein lipase Biol. Chem. 1994; Scholar) because are on binding of MAb 5D2 to LPL not H. T. J. M. Schotz M.C. Lipoprotein lipase Biol. Chem. 1994; Scholar) because the for to mainly or in the amino terminal are of LPL of MAb 5D2 H. T. J. M. Schotz M.C. Lipoprotein lipase Biol. Chem. 1994; Scholar) is to interaction with of the located binding in both MAb 5D2 not of LPL for H. T. J. M. Schotz M.C. Lipoprotein lipase Biol. Chem. 1994; Scholar) because the is from the active site and its on the and of the of MAb have the used are to experimentally or this for 5D2 are to and for of the is This study a from the to the and a the The is the and the for hepatic triglyceride lipase lipoprotein lipase monoclonal antibody lipase