Biochimica et Biophysica Acta, 1128 (1992) 113-115 © 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2760/92/$05.00

BBALIP 50342

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Rapid Report

Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity Ming-Sun Liu a, Yuanhong Ma a, Michael R. Hayden a and John D. Brunzell b " Department of Medical Genetics, Unil'ersity of British Cohonbia, Vancom,er (Canada) and t, Department of Medicine, Unil,ersity of Washington, Seattle, WA (USA) (Received 6 July 1992)

Key words: Lipoprotein lipase; Monoclonal antibody; Epitope mapping

A monoclonal antibody, 5D2, which inhibits human iipoprotein lipase (hLPL) activity has been widely used for assessment of LPL immunorcactive mass in the clinical evaluation of patients [I] and for analysis of structure-function relationships of LPL [2,3]. We have mapped the epitope on LPL, recognized by the 5D2 antibody, within residues 396-405. Ala4°qjis the critical amino acid residue conferring epitope specificity. This knowledge confirms that the C-terminal domain of LPL plays a critical role in LPL activity and also provides important information liar studies exploring the structure-function relationship of LPL using this antibody.

Lipoprotein lipase (EC 3.1.1.34) is a major enzyme in lipid metabolism which is active-bound to the heparan sulfate proteoglycan of the endothelial cell, where it hydrolyses the triacyiglycerol core of circulating chylomicrons and very-low-density lipoprotein (VLDL), generating free fatty acids and lipid remnants [4]. The cDNAs of LPL of several species have been cloned and sequenced [5-9]. The human eDNA encodes a total of 448 amino acid residues which constitutes the mature protein which is highly homologous to LPL of other species [6]. LPL also has significant homology with hepatic (HL) and pancreatic lipase (PL) which are thought to be members of the same gene family [ 10]. A number of polyclonal and monoclonal antibodies have been developed to detect LPL immunoreactive mass and for assessment of its function [1,11-16]. A monoclonai antibody, named 5D2, was generated against bovine LPL and found to be fully reactive to human LPL [1]. This antibody has been shown to completely inhibit lipase activity and has been widely used for assessment of LPL immunoreactive mass in many patients with defective LPL activity who have mutations in the LPL gene [1,3,17-28]. Recently, this 5D2 antibody has also been used for the analysis of structure-function relationship of LPL by assessment of HL-LPL chimeric proteins [2].

Correspondence to: M.R. Hayden, Rm 416, NCE Building, 2125 East Mall, Vancouver, B.C., V6T I Z4, Canada.

In an effort to further study structure and function of LPL, it is important to determine the epitope on LPL which is recognized by the 5D2 antibody. In these experiments, we have used the homology between different LPL species together with the fact that the 5D2 antibody only recognizes LPL of certain species, to locate the epitope specific for 5D2. Post-heparin plasma from human, mouse, cat and chicken were collected 10 min after intravenous heparin injection (60 IU/kg). LPL is released into plasma from the surface of the endothelial cell after heparin injection. Blood was initially placed on ice and plasma was separated by centrifugation (3000 g 10 rain at 4°C) and samples were frozen immediately at -70°C until analysis. LPL of post-heparin plasma from human, cat, chicken and mouse were assessed by a sandwich ELISA using the 5D2 antibody as probe. A sandwich ELISA for the detection of LPL nondenaturcd dimer was performed as described previously [1] with some modifications. Briefly, the 5D2 antibody was coated on a microtiter well (Maxisorp, Nunc) at 37°C for 4 h. The coated plates were then rinsed with phosphate buffered-saline (PBS) containing 0.05% Tween-20 (PBST). The plasma from human, mouse, cat and chicken was added into 5D2 antibody coated microtitcr plates at 4"C overnight. After rinsing with PBST, the plates were incubated with 5D2 antibody conjugated with horseradish peroxidase (5D2-HRP) for 3 h at room temoerature. The plates were finally rinsed with PBST and the peroxidase activity was measured by

114 absorbance at 450 nm using 3,Y,5,5'-tetramethyl benzidine (Sigma, St Louis, MO) as a substrate. The 5D2 antibody was found to recognize LPL protein in human, cat and chicken post-heparin plasma, but did not recognize LPL in mouse post-heparin plasma. Human and mouse LPL have 93.5% homology. Two regions involving residues 325-333 and 396-405 were detected which showed only 61.5% and 60% homology, respectively, between human and mouse LPL compared to 95% in the rest of the protein. We postulated that amino acid sequences within these two regions might contribute to epitope specificity, Therefore, two oligopeptides were synthesized based on the sequences of these two regions in human LPL for further analysis. The amino acid sequence of these two synthetic oligopeptides are TESETHTNQ (amino acid residue 325-333 in human LPL; named HLPL-1) and SPGFAIQKIR (amino acid residue 396405 in human LPL; named HLPL-2), respectively. These two synthetic oligopeptides were dissolved in 50 ttl of 20% sodium dodecyl sulfate (SDS) solution then diluted and coated on microtiter plates, respectively. The coated plates were rinsed with PBST. The 5D2HRP was ~.hen added to the plates for 3-h incubation at room temperature. The peroxidase activity measurement was performed as described above. Fig. 1 shows the binding of 5D2 antibody to HLPL-1 and HLPL-2 oligopeptides. The 5D2 antibody was found to react with the HLPL-2 but not with the HLPL-I. This suggested that epitope specific residues a~e within HLPL-2, Also, it is noteworthy that the epitope for 5D2 antibody is not destroyed in 20% SD$ solution which suggests that this epitope is sequential and continuous, 'Fo assess the essential residues for epitope specificity, we compared the HLPL-2 corresponding sequences, of cat (unpublished data), bovine [7], chicken [9], human [6] which are recognized by the 5D2 antibody and those of mouse [5] and human HL [29] which are not recol;nized by 5D2 antibody [I] (Fig. 2). Mouse LPL and human HL shares at least one residue with

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LOG (ANTIBODYDILUTION) Fig. I. Bindm~ of the 5D2 antibody to HLPL-i (e) and HLPL-2 (o) synthetic oligopeptides.

Enzyme

Amino

acid resxaue

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405}

5D2 c e a c u i o n

human

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bovine

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chicken

LPL

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mouse

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I

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human

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Fig. 2. Comparison of the amino acid sequence of HLPL-2 corresponding to residues 396-405 in hLPL and equivalent residues in bovine, cat, chicken and mouse LPL and human HL. The amino acid residues corresponding to residue 400 of hLPL of different species arc boxed. The cross-reactivity of 5D2 antibody to these sequences is indicated on the right.

one of the other species of LPL at all the residues between 396-405 except that which is equivalent to residue 400 in human LPL. This comparison therefore revealed that the residue 4(~L which is encoded by exon 8 of the LPL gene, is likely to be critical for epitope specificity. The epitope is retained when the residue changes from alanine (human LPL) to aspartic acid (bovine LPL) or to threonine (cat and chicken LPL), but not to valine (mouse LPL). Single amino acid substitutions, altering the epitope specificity, have also been reported in the studies of apolipoprotein B (apo B) [30]. in this particular instance, a similar change of the GCT codon for Ala-591 to a GCT codon for Val-591 also abolished epitope specificity for a specific monoclonal antibody. It has previously been shown that the 5D2 antibody inhibits lipase but not esterase activity [2]. The mechanism of inhibition of lipasc activity by 5D2 is uncertain. However, it has been suggested that 5D2 could either dissociate the active dimer form of LPL or affect large lipid substrate binding to LPL or prevent it reaching the catalytic site [2]. Based on the efficacy of the sandwich ELISA system for detection of LPL dimer, it is unlikely that the binding of 5D2 antibody to LPL protein destroys dimerization. We have shown that the epitope recognized by 5D2 antibody is clearly located near the end of the C-terminal domain distant from the catalytic Asp-156-His241-Ser-132 triad [28], the proposed lipid binding site (residues 105-209) [31] and the apolipoprotein CII (apo Cli) binding site [2] in the LPL protein which are predicted to lie in the N-terminal domain [31]. Even though residues known to directly participate in the catalytic triad are in the N-terminal domain of the protein, this antibody might affect lipase activity as a result of particular conformational changes in the tertiary structure of LPL which affect either the lipase binding site or the catalytic triad. Knowledge of the atomic structure of this protein would provide addi-

115

tional information which would enhance understanding concerning the interaction of different residues in the LPL, whatever the precise mechanism for inhibition of lipase activity. These experiments have confirmed that residues within C-terminal domain of LPL may also play a critical role in LPL activity. This work was supported by the British Columbia Heart Foundation, The Medical Research Council of Canada and NIH grant DK02456. Dr. Ming-Sun Liu is a Merck Frosst/Canada Genetic Disease Network post-doctoral fellow, Dr. Yuanhong Ma is a MRC (Canada) post-doctoral fellow and Dr. Michael Hayde~i is an established investigator of the British Columbia Children's Hospital. References I Babirak, S.P,, Iverius, P-H., Ftkjimoto, W.Y. and Brunzell, J.D. {1989) Arteriosclerosis 9, 326-334. 2 Wong, H,, Davis, R.C,, Nikazy, J,, Seebart, K,E. and Schotz, M.C. (19ql) Proc, Natl, Acad. Sci. USA 88, 112911-11294. 3 Peterson, J.r Fujimoto, W.Y. and Brunzell, J.D. (1992) J. Lipid Res., in press. 4 Brunzell, J.D. {1989) in The Metabolic Basis of Inherited Disease. 6th Edn. (C. Striver, A.I. Beaudet, Sly, W.S., eds.), pp. 1165-1180, McGraw-Hill, New York. 5 Kirchgessner, T.G., Svenson, K.L., Lusis, A.J. and Schotz, M.C. (1987) J. Biol. Chem. 262, 8463-8466. 6 Wion, K.L., Kirchgessner, T.G., Lusis, A.J., Schotz, M.C. and Lawn, R.M. (1987) Science 235, 1638-1641. 7 Senda, M., Oka, K., Brown, W.V., Qasba, P.K. and Furuichi, Y. (1987) Proc. Nail. Acad. Sci. USA 84, 4396-4373. 8 Enerback, S., Scrub, S.H., Bengtsson-Olivecrona, G., Carlsson, P., Hermansson, J.S., Olivecrona, T. and Bjarsell, G. (1987) Gene 58, 1-12. 9 Cooper, D.A., Stein, J.C., Strieleman, P.J. and Bensadoun, A. (1989) Biochim. Biophy:,. Acta 1008, 92-101. 10 Komaromy, M.C. and Schotz, M.C. (1987) Pro¢. Natl. Acad. Sci. USA 84, 1526-1530. II Shirai, K., Wisher, D.A., Johnson, JD., Srivastava, L.S. and Johnson, R.L. (1982) Biochim. Biophys. Acta 712, 111-211. 12 Olivecrona, T. and Bengtsson, G. (1983) Biochim. Biophys. Acta 752, 38-45. 13 Socorro, L. and Johnson, R.L. (1985)J. Biol. Chem. 260, 63246328.

14 Goers, J.F., Pete[sen, M.E,, Kern, P.A., Ong, J. and Schotz, M.C. (1987) Anal. Biochem. 166, 27-35. 15 Vannier, C., Deslex, S,, Pradines-Figueres, A. and Aiihaud, G. (1989) J. Biol. Chem. 264, 13199-13205. 16 lkeda, Y., Takagi, A., Ohkaru, Y., Nogi, K., lwanaga, T., Kurooka, S, and Yamamoto, A. (1990)J. Lipid Res. 31, 1911-1924. 17 Auwerx, J.H., Babirak, S.P., Fujimoto, W.Y., lverius, P.H. and Brunzell, J.D. (1989) Eur. J. Clin. Invest. 19, 433-437. 18 Devlin, B., Deeb, S., Brunzell, J.D., Hayden, M.R. (1990) Am. J. Hum. Genet. 46, 112-119. 19 Monsalve, M.V., Henderson, H., Roederer, G., Julien, P., Deeb, S., Kastelein, J,J.P., Peritz, L., Devlin, R., Bruin, T,, Ven Murthy, M.R., Gagne, C., Davignon, J., Lupien, P.J., Brunzell, J.D. and Hayden, M.R. (1990) J. Clin. Invest. 86, 728-734. 20 Auwerx, J.H,, Babirak, S.P., Hokanson, J.E., Stahnke, G., Will, H., Deeb, S.S. and Brunzell, J.D. (1990) Am. J. Hum. Genet. 46r 4711-477, 21 Beg, O.U., Meng, MS., Skarlatos, S.I., Previato, L., Brunzcll, J.D., Brewer, H.B. and Fojo, S.S. (1990) Proc. Natl Acad. Sci. USA 87, 3474-3478. 22 Peritz, L.N., Brunzell, J.D., Harvey-Clarke, C., Pritchard, P.H., Jones, B.R. and Hayden, M.R. (19911) Clin. Inve~t. Mcd. 13. 259-263. 23 Henderson, lt.E., Ma, Y., Hassan, M.F., Monsalvc, M.V., Winkler, F., Guber.mlor, K., Marais, A.D., Brunzcll, J.l). and Ilayden, M.R. (1991)J. Ciin. Invest. 87, 21}05-2011. 24 Ma, Y., Henderson, H.E., Ven Murthy, M.R., Rocdercr, G., Monsalve, M.V., Clarke, L.A., Julien, P., Gagne, C., Davignon, J., Lupien, P., Brunzell, J.D. and Hayden, M.R. (1991) N. Engl. J. Med. 324, 1761-1767. 25 Dichek, H.L., Fojo, S.S., Beg, O.U., Skarlatos, S.I., Brunzell. J.D., Cutler, G.B., Jr. and Brewer, H.B., Jr. (1991) J. Biol. Chem. 266, 473-466. 26 Henderson, H.E., Devlin, B., Peterson, J., Brunzell, J.D. and Hayden, M.R. (1991)Mol. Biol. Med. 7, 511-517. 27 M,Y,, Bruin, T., Tuzgol, S., Wilson, B.I., Roedercr, G., Liu, M.S.. Davignon, J., Kastelein, J.J.P., Brunzell, J.D. and Hayden, M.R. (1992) J. Biol. Chem. 267, 1918-1923. 28 Emmerich, J., Beg, O.IJ., Peterson, J., Prcviato, S., Brunzcll, J.D., Brewer, H.B. Jr. and Fojo, S.S. (1997) J. Biol. ('hem. 267, 4161-41h5. 29 Dalta, S., You, C.-C., Li, W.-II., Van Tuincn, P., l,:,dbcttcr, D.iI., Brown, M.A., Chen, S.-H., Liu, S.-W. and Chan. L. (1988) J. Biol. Chem. 263, 1!1)7-1110. 31) Wang, X., Schlapfer. P., Ma, Y., Butler, R., Elovson, J. and Schumaker, V.N. (1988) Arteriosclerosis 8, 429-435. 31 Persson, B., Bengtsson-Olivecrona, G., Enerback, S., Olivecrona, T. and Jornvall, H. (1989) Eur..I. Biochem. 179, 39-45.

Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity.

A monoclonal antibody, 5D2, which inhibits human lipoprotein lipase (hLPL) activity has been widely used for assessment of LPL immunoreactive mass in ...
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