Ciencia y Tecnología Alimentaria Sociedad Mexicana de Nutrición y Tecnología de Alimentos
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ISSN (Versión impresa): 1135-8122 ISSN (Versión en línea): 1696-2443 MÉXICO
1998 J. A. Ramírez / E. Bandman / M. Wick / M.O. Martín Polo MONOCLONAL ANTIBODIES AS A TOOL FOR STUDYING THE PROTEINSTRUCTURE RELATIONSHIP IN FISH MYOSIN Ciencia y Tecnología Alimentaria, julio, año/vol. 2, número 001 Sociedad Mexicana de Nutrición y Tecnología de Alimentos Reynosa, México pp. 6-11
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal Universidad Autónoma del Estado de México http://redalyc.uaemex.mx
Cienc. Tecnol. Aliment. Vol. 2, No. 1, pp. 6-11, 1998 Copyright 1998 Asociación de Licenciados en Ciencia y Tecnología de los Alimentos de Galicia (ALTAGA).
ISSN 1135-8122
MONOCLONAL ANTIBODIES AS A TOOL FOR STUDYING THE PROTEIN-STRUCTURE RELATIONSHIP IN FISH MYOSIN ANTICUERPOS MONOCLONALES COMO HERRAMIENTA PARA EL ESTUDIO DE LA RELACIÓN PROTEÍNA-ESTRUCTURA EN MIOSINA DE PEZ
ANTICORPOS MONOCLONAIS COMO FERRAMENTA PARA O ESTUDO DA RELACIÓN PROTEÍNAESTRUCTURA EN MIOSINA DE PEIXE
Ramírez, J. A.1*, Bandman, E.2, Wick, M.2, Martín-Polo, M.O.1 1
Facultad de Química, Universidad Autónoma de Querétaro, México. Dept. of Food Science and Technology, Univ. California, Davis. USA.
2
* Autor para la correspondencia. Dirección para la correspondencia: Dr. J. A. RAMIREZ. Unidad Académica Multidisciplinaria Reynosa Aztlán. Universidad Autónoma de Tamaulipas. Calle 16 y Fuente de Diana. Col. Aztlán 88740 Reynosa, Tamaulipas, México.
E-mail:
[email protected]
Abstract Fish muscle protein presents unique properties such as reduced frozen storage stability and setting phenomena. Both two are associated with myosin interactions and are highly dependent on fish specie. Although protein functional properties are highly correlated with amino acid sequence and with distinct structural domains, it is very difficult to determine this relationship for fish myosin, due to its high molecular weight and the great varieties of fishes. The present study deals with the feasibility for studying such relationship employing available isolated monoclonal antibodies whose epitopes has been well-characterized on chicken myosin heavy chain. This alternative opens a new way to be explored. Keywords: immunoassay, monoclonal antibodies, myosin, fish.
Resumen La proteína de los peces presenta propiedades únicas, tales como la reducida estabilidad al almacenaje en congelación y el fenómeno de asentamiento. Ambas están asociadas con interacciones de la miosina y son altamente dependientes de la especie de pez. Aunque las propiedades funcionales de las proteínas están muy relacionadas con la secuencia de aminoácidos y con distintos dominios estructurales, es muy difícil determinar su relación para la miosina de la proteína de los peces, debido a su alto peso molecular y la gran variedad de peces. El presente estudio trata sobre la viabilidad de estudiar esa relación empleando anticuerpos monoclonales aislados y disponibles comercialmente, cuyos epitopes han sido caracterizados para la cadena pesada de miosina de pollo. Esta alternativa abre una nueva vía para ser explorada. Palabras clave: immunoensayo, anticuerpos monoclonales, miosina, peces.
Resumo A proteína dos peixes presenta propiedades únicas, tales como a reducida estabilidade á almacenaxe por conxelación e ó fenómeno de asentamento. Ambas están ligadas a interaccións da miosina e son moi dependentes da especie de peixe. Ainda que as propiedades funcionais das proteínas están moi relacionadas ca secuencia de aminoácidos e cos dominios estructurais, é moi difícil determiñar a sua relación ca miosina da proteína dos peixes, debido ó seu alto peso molecular e a gran variedade de peixes. O presente estudio trata de evaluar a viabilidade de estudar esa relación empregando anticorpos monoclonais aillados e dispoñibels comercialmente, cuns epitopes que foron caracterizados para a miosina do polo. Esa alternativa abre unha nova vía para ser explorada. Palabras clave: immunoensaio, anticorpos monoclonais, miosina, peixes.
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INTRODUCTION
reported to be important to the structure-function relationship (Damodaran, 1994). However, fish myosins have not yet being studied to determine their primary structure. This may be due to the number of fish species, the high molecular weight of this protein and the fact that the determination of amino acid sequence is not an easy task. Therefore, immunochemistry, using monoclonal antibodies has been an alternative to demonstrate differences among myosin heavy chains (MYHCs) isoforms in the same muscle and for comparison among different MYHC isoforms in distinct species.
Myofibrillar proteins are responsible for the functional properties such as gelling, water holding capacity and binding in animal meat products. Despite the similar role on functionality, fish muscle proteins exhibit unique functional properties. The principals are reduced frozen storage stability and setting phenomena (Toyoda et al., 1992; MacDonald and Lanier, 1991). The latter is the ability of fish myofibrillar proteins to gel between 5 and 43 °C, as a function of incubation time, after solubilization with 2-3% of salt (Toyoda et al., 1992). Recently it has been proposed that such differences could be attributed to the fact that fish are cold-blood animals while poultry and mammals are warm-blood (Toyoda et al., 1992). Moreover, some fish species are more susceptible to frozen instability than others. Setting phenomena is correlated with different rates and optimal temperatures depending on the species (Kamath et al., 1992). The instability during frozen storage has been associated with a lose of stability of fish myosin (the major contractile protein of skeletal muscle), and is dependent on habitat temperature, with myosin from cold-water species being less stable than myosin from warm-water species (Davies et al., 1994). On the other hand, the unique setting phenomena observed in fish myofibrillar proteins is also associated with myosin interactions (Nowsad et al., 1992). Although myosin has been implicated in both properties, it remains unclear if there is a structure-function correlation between myosin from different fish species and functionalityproperties.
Several studies have shown that monoclonal antibodies that react with chicken myosins also reacted with myosins from other animals. The epitope for the 5C3 antibody has been reported to be present in rabbit psoas muscle which contains fast skeletal MYHC isoforms (Winkelmann and Lowey, 1986), and F59 reacts with the S-1 from different vertebrate MYHC isoforms such as, chicken, quail, rat, rabbit, turtle, newt, frog, goldfish, electric ray and shark, but did not react with nematode, slime mold and amphioxus the invertebrate MYHCs (Miller et al., 1989). On the other hand the 10F12 and 12C5 chicken myosin antibodies did not react with rabbit myosin from psoas muscle (Winkelman and Lowey, 1986). Other antibodies, not used in this research, like F18, F27, F30 and F49, whose epitopes are present on chicken myosin S-1, reacted with other vertebrates, such as quail, rat and rabbit (Miller et al., 1989).
Immunoassays employing specific monoclonal antibodies have been used to: 1) determine evolutionary relationships among myosin from different species (Miller et al., 1989), 2) determine solubility properties (Wick et al., 1996), 3) identify different chicken skeletal myosin isoforms present in embryonic, neonatal, adult fast, slow and gizzard muscle (Miller et al., 1989).
The objective of this work was to determine if fish skeletal muscle myosin from Tilapia nilotica shares some of the epitopes observed on chicken skeletal muscle myosins. The seven specific monoclonal antibodies employed on this work, were selected because their epitopes have been well ubicated on the chicken myosin (Table 1), and so, if they are present in the same fish myosin regions, they could be an excellent tool to study differences on functional properties among fish species, associated with differences on protein structure.
There are different myosin isoforms in vertebrates that are recognized by specific monoclonal antibodies, indicating differences in protein primary structure. Chicken fast myosin isoforms, present mainly in white muscle, and slow myosin isoforms present in red muscle, differ in ATPase activity (Moore et al., 1992), thermal properties (Xiong et al., 1987), gelling capacity (Xiong, 1994), amino acid sequence and epitopes for specific monoclonal antibodies (Moore et al., 1993; Bandman, 1992).
MATERIAL AND METHODS Myosin extraction and purification Alive Tilapia nilotica fishes were obtained from a Querétaro, México dam. Myosin was extracted from 30 g of sample obtained from several fishes, according to Martone et al. (1986). The extracted myosin was solubilized in 0.6 M KCl, 20 mM Tris-HCl (pH 7.0) and stored for 7 days at 4 °C because to transportation requirements. Subsequently, samples were dialyzed against 80 mM sodium pyrophosphate, 2 mM magnesium chloride, 2 mM EGTA, pH 9.5 buffer, after which glycerol was added to make an 1:1 glycerol-buffer solution and stored for 3 weeks at -20 °C. Myosin was further purified by ion exchange chromatography as described by Richards et al. (1967). The entire process was performed at 4 °C.
Vertebrate myosins present similar structure and functional properties. However some functional properties vary among species and the origins being unclear. It is possible that amino acid sequence more than amino acid composition is the answer. The amino acid sequence dictates the three-dimensional structure of a protein and therefore its thermodynamic stability, charge distribution, hydrophilic and hydrophobic characteristics, and has been
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Table 1.Reported specificity and epitopes localization for chicken skeletal muscle myosin antibodies.
ANTIBODY
SPECIFICITY
FRACTION
LOCALIZATION (RESIDUES)
DISTANCE (nm)
5C3
Adult MYHC fast isoforms [1, 2]
LMM
----
129.29
NA4
All sarcomeric MYHC isoforms [3-4]
LMM
994-1004
125.69
Embryonic and adult MYHC fast isoforms [3–4]
LMM
804-810
55.77
Adult MYHC isoforms [3-4]
LMM
428-470
46.12
EB165 AB8 10F12
Adult MYHC fast isoforms [5]
S-2
----
---
12C5
Adult and embryonic MYHC fast isoforms [2, 5–6]
S-1
29-60
14.00
F59
All sarcomeric fast MYHC isoforms [1]
S-1
211-231
---
Localization: From NH2 terminus to COOH terminus. Distance: From the myosin head/rod junction. (1) Miller et al., 1989; (2) Winkelmann et al., 1983; (3) Wick et al., 1996; (4) Moore et al., 1993; (5) Winkelmann and Lowey, 1986; (6) Winkelmann et al., 1993. -- Data not reported in reference
Figure 1. Illustration of the sites where monoclonal antibodies bind to chicken skeletal myosin. Distances showed are according with the localization of residues indicated on Table 1, except for 5C3 and 10F12. (Adapted from Wick et al., 1996 ).
Purity of the selected fractions was confirmed by SDSPAGE (7% T). Fractions were concentrated by dialysis against glycerol.
transfers were incubated with monoclonal antibodies at various dilutions (i.e. 1:500 or 1:1000) for 60 min at room temperature, in phosphate buffered saline pH 7.4, 5% nonfat dry milk (PBSM).
Electrophoresis and Immunoblotting NA4, 5C3, EB165 and AB8 antibodies were raised at the Department of Food Science and Technology, University of California at Davis. F59 was supplied by Frank E. Stockdale from the Department of medicine, Stanford University School of Medicine, Stanford, Ca. 12C5 and 10F12 were supplied by Susan Lowey from Department of Biochemistry, Rosenstiel Basic Medical
Protein samples were prepared for electrophoresis by mixing protein solution (0.2 to 0.5 mg/mL) with 5X sample buffer and boiling the sample for 3 min. SDSPAGE was performed according to Laemmli (1970). After electrophoresis, proteins were transfered to nitrocellulose paper as described by Bandman and Zdanis (1988). The
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(A) 5C3
(B) NA4
(C) EB165 -MYOSIN -ROD
-LMM
H
M
S-1 R-L H
M
S-1
R-L
H M
S-1
R-L
Figure 2. Localization of epitopes on fish skeletal myosin fragments (rod, LMM, S-1), for 5C3, NA4 and EB165 chicken skeletal myosin monoclonal antibodies. (H) High range prestained molecular weight standard; (M) Myosin; (S-1) Purified S-1 fraction; (R-L) Purified rod and LMM fractions.
Fig. 1. Monoclonal antibodies, whose epitopes have previously been reported to reside in LMM domain in chicken myosin, reacted with both LMM and rod fractions in fish myosin (see Fig. 2). Antibodies whose epitopes were located in the S-2 domain reacted only with the rod in fish myosin (Fig. 3).
Sciences Research Center, Brandeis University. Waltham, Massachusetts. Preparation of S1, light meromyosin (LMM) and rod fraction S-1 was prepared according to Margossian and Lowey (1982). Purity was determined by SDS-PAGE. The S1 fraction was concentrated by dialysis in glycerol. The final solution of S1 was obtained by dialysis against 0.6 M KCl, 0.15 M potassium phosphate (K2HPO4), at 4 °C overnight. Despite results reported with rabbit, in fish myosin EDTA did not protect the neck region and the described proceeding resulted in the production of S-1, LMM and rod simultaneously. After removal of S1, the insoluble precipitate (containing myosin, rod and LMM) was dispersed in 0.6 M KCl, 0.15 M potassium phosphate, pH 7.0, and rod and LMM fractions were isolated according to Margossian and Lowey (1982). A 0.6M KCl, 0.15 M potassium phosphate buffer, pH 9.5, 0.001 M DTT was employed instead of 0.03 M KCl, 0.01 M potassium phosphate buffer, pH 7.0 recommended. Under these conditions, much of the LMM and rod were solubilized, whereas the denatured myosin, which remained insoluble, along with insoluble rod and LMM was removed by centrifugation. Purity was determined by SDS-PAGE. Both fractions were pooled and concentrated by dialysis against glycerol. The final solution of rod and LMM was obtained by dialysis against 0.15 M potassium phosphate (K2HPO4), 0.6 M KCl at 4 °C overnight.
Five of the seven chicken myosin monoclonal antibodies reacted with the fish myosin: 5C3, NA4, EB165, 10F12 and F59 (Fig. 2 and 3). The AB8 and 12C5 antibodies did not react with fish myosin. 5C3, NA4 and EB165 monoclonal antibodies, reacted with both fish myosin rod and LMM, but not with S-1 (Fig. 2). These results indicate that their epitope must be on the LMM fraction of fish myosin, which is similar to chicken myosin. The NA4 gave a special increase on sensitivity, showing a greater background, detecting fractions of rod in purified S-1 fractions, which were not detected by electrophoresis. The 10F12 antibody stained only the band corresponding to the myosin rod fraction (Fig. 3A). The F59 monoclonal antibody reacted with the fish myosin S-1 preparation (Fig. 3B), but did not react either with rod or LMM indicating that the F59 epitope is located on the head domain of fish myosin. These results indicate that the five monoclonal antibodies, which reacted with fish skeletal muscle myosin, did so with the same myosin regions reported for chicken skeletal myosin. Because monoclonal antibodies only react with the identical primary sequence, there is likely that epitopes are in the same location (Fig. 1). The discrepancies in the location of the antibodies are a common occurrence (Wick et al., 1996). The position of the AB8 antibody visualized on the rod (Table 1) was consistent with the position of the antibody epitope as determined by deletion set mapping (Moore et al., 1992). The position of EB165 antibody visualized by electron microscopy was closer to the N-terminus of the LMM than predicted by deletion set mapping. However, others authors have reported
RESULTS AND DISCUSSION Purified fish myosin rod, LMM and S-1 preparations were employed to determine the location of the epitope for each monoclonal antibodies. The specificity of the monoclonal antibodies employed in this study are indicated in Table 1. Their epitopes are found in different regions of the MYHC: LMM, S-2 and S-1 as is showed in
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(A) 10F12
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(B) F59 -MYOSIN - ROD - S-1
H
M
S-1
R-L
H
M
S-1
R-L
Figure 3. Localization of epitopes on fish skeletal myosin fragments (rod, LMM, S-1), for 10F12 and F59 chicken skeletal myosin monoclonal antibodies. (H), (M), (S-1) and (R-L) as in Fig. 2.
similar discrepancies with antibodies that bound near the C-terminus of the myosin rod (Rimm et al., 1990). On Figure 1, the epitopes were ubicated according with their position determined by deletion set mapping (Table 1).
studies, and accordingly with this results they could be employed to determine the existence of structurefunctionality relationship for fish myosins. REFERENCES
Our results demonstrate that five of the seven monoclonal antibodies studied, reacted with the same domains in chicken and fish MYHC. 5C3, NA4 and EB165 epitopes were located in the LMM of fish MYHC, likewise, F59 reacted with S-1 and 10F12 only reacted with the S-2 fractions in both fish and chicken MYHC. The reactivity of fish skeletal myosin to chicken skeletal myosin monoclonal antibodies, and the localization of epitopes on the same myosin fraction, might be indicative of an evolutionary relationship between these two species: chicken and Tilapia nilotica.
Bandman, E. 1992. Contractile protein isoforms in muscle development. Dev. Biol. 154, 273-283. Bandman, E. and Zdanis, D. 1988. An immunological method to assess protein degradation in post-mortem muscle. Meat Sci. 1-19. Damodaran, S. 1994. Structure-function relationship of food proteins. In Protein functionality in food systems. (Edited by Hettiarachchy, N.S. and Ziegler, G.R.), pp 1-37. IFT Basic Symposium Series. Marcel Dekker, Inc.
The feasibility of employing monoclonal antibodies to detect important domains for functional and/or structural properties, has been showed by Wick et al. (1996), who found some evidence about the participation of the NA4 and 5C3 antibodies on the solubility of myosin in vitro at low ionic strength. Although EB165 and AB8 antibodies did not affect the solubility properties, or the morphology of myosin aggregates formed in low salt conditions it was suggested that domains responsible for these functions may be located outside the LMM domain. However preliminary studies could not to associate such antibodies with frozen storage stability of fish myosin obtained from Tilapia nilotica (data not shown).
Davies, J.R., Ledward, D.A., Bardsley, R.G. and Poulter, R.G. 1994. Species dependance of fish myosin stability to heat and frozen storage. Int. J. Food Sci. Technol. 29, 287-301. Kamath, G.G., Lanier, T.C., Foegeding, E.A. and Hamann, D.D. 1992. Nondisulfide covalent cross-linking of myosin heavy chain in «setting» of Alaska pollock and atlantic croaker surimi. J. Food Biochem. 16, 151-172. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680.
Although abundant information exists concerning the different functional properties among fish species, the cause of such differences remains unclear. Because myosin is considered the principal protein responsible of the functional properties from different vertebrate muscles, it is possible that the use of monoclonal antibodies can be employed to correlate functionality and myosin structure from different species. A great advantage for food scientists is the existence of isolated monoclonal antibodies that are being employed for evolutionary relationship
Macdonald, G.A. and Lanier, T.C. 1991. Carbohydrates as cryoprotectants for meats and surimi. Food Technol. 45, 150-159. Margossian, S.S. and Lowey, S. 1982. Preparation of myosin and its subfragments from rabbit skeletal muscle. Meth. Enzymol. 85, 55-71.
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Martone, C.B., Busconi, L., Folco, E.J., Trucco, R.E. and Sánchez, J.J. 1986. A simplified myosin preparation from marine fish species. J. Food Sci. 51, 1554.
Toyoda, K., Kimura, I., Fujita, T., Noguchi, S.F. and Lee, C.M. 1992. The surimi manufacturing process. In Surimi Technology (Edited by T.C. Lanier and C.M. Lee), pp 79-112. Marcel Dekker, Inc.
Miller, J.B., Teal, S.B. and Stockdale, F.E. 1989. Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression. J. Biol. Chem. 264, 13122-13130.
Wick, M., Tablin, F. and Bandman, E. 1996. The effects of Anti-LMM antibodies on the solubility of chicken skeletal muscle myosin. J. Food Biochem. 20: 379-395.
Moore, L.A., Arrizubieta, M.J., Tidyman, W.E., Herman, L.A. and Bandman, E. 1992. Analysis of the chicken fast myosin heavy chain family. Localization of isoform-specific antibody epitopes and regions of divergence. J. Mol. Biol. 225, 1143-1151.
Winkelmann, D.A. and Lowey, S. 1986. Probing myosin head structure with monoclonal antibodies. J. Mol. Biol. 188, 595-612. Winkelmann, D.A., Kinose, F. and Chung, A.L. 1993. Inhibition of actin filament movement by monoclonal antibodies against the motor domain of myosin. J. Muscle Cell Motil. 14, 452-467.
Moore, L.A., Tidyman, W.E., Arrizubieta, M.J. and Bandman, E. 1993. The evolutionary relationship of avian and mammalian myosin heavy-chain genes. J. Mol. Evol. 36, 21-30.
Winkelmann, D.A., Lowey, S. and Press, J.L. 1983. Monoclonal antibodies localize changes on myosin heavy chain isozymes during avian myogenesis. Cell 34, 295-306.
Nowsad, A.A.K.M., Kanoh, S. and Niwa, E. 1992. Electrophoretic behavior of cross-linked myosin heavy chain in suwari gel. Nippon Suisan Gakkaishi. 59, 667671.
Xiong, Y.L. 1994. Myofibrillar protein from different muscle fiber types: implications of biochemical and functional properties in meat processing. CRC Crit. Rev. Food Sci. Nutr. 34, 293-319.
Richards, E.G., Chung, C.S., Menzel, D.B. and Olcott, H.S. 1967. Chromatography of myosin on diethylaminoethyl-sephadex A-50. Biochemistry 6, 528-540.
Xiong, Y.L., Brekke, C.J. and Leung, H.K. 1987. Thermal denaturation of muscle proteins from different species and muscle types as studied by differential scanning calorimetry. Can. Inst. Food Sci. Technol. J. 20, 357362.
Rimm, D.L., Kaiser, D.A., Bhandari, D., Maupin, P., Keiart, K. and Pollard, T.D. 1990. Identification of functional regions on the tail of Acanthamoeba myosinII using recombinant fusion proteins. I. High resolution epitope mapping and characterization of monoclonal antibody binding sites. J. Cell Biol. 11, 2405-2416.
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