Associate-Professor Clive Evans

Associate-Professor

Molecular, Cellular & Developmental Biology

Phone: 09-3737599 x87245
Rm 144
Email: c.evans@auckland.ac.nz

Calpains | Fish Antifreeze | Ecotoxicology and Disease

The Clive Evans group is concerned with the analysis of the expression patterns of different calpains during development, using the zebrafish and the mouse as model systems. The laboratory has identified a number of splice variants of calpain 3 that are differentially expressed during the differentiation of muscle cells from myoblasts to myofibres.

Further analysis of these genes will involve sequencing and characterising the expression patterns of the different orthologous genes, and the generation of a phylogenetic gene map for the calpain family. Finally, environmental toxicology also provides a research focus for the Evans laboratory. This focus involves a study of the impact of organic pollutants in Antarctica.

Calpains

Our laboratory has a number of interests which focus on different aspects of normal or perturbed development. One relates to the function of calpains in development and disease, another to the development of antifreeze in Antarctic fishes and a third is in the area of Molecular Ecotoxicology.

A number of different research projects focussing on calpains in mammalian and zebrafish experimental systems are available in the Evans laboratory. Please contact us if you wish to discuss things further and receive more specific details.

1. Calpains in Development and Disease

The calpains (EC 3.4.22.17) are a family of non-lysosomal, Ca2+-dependent neutral cysteine proteases which have been implicated in the regulation of a number of cellular processes including signal transduction, cytoskeletal dynamics and muscle homeostasis. Two calpains (calpains -1 and-2) are distributed ubiquitously whereas the others identified to date are confined to just one or a few cell lineages (Sorimachi, Ishiura & Suzuki, 1997).

Skeletal muscle calpains

Adult mammalian skeletal muscle contains three calpain isoforms (calpains -1, -2 and -3), while a fourth (calpain-6) is found in skeletal muscle only in the embryo where its expression domain closely overlaps that of calpain-3 (Dear & Boehm, 1999).

The ubiquitous calpains exist as heterodimers with the large (80 kD) subunit retaining enzymatic activity when it is dissociated from its 30 kD regulatory partner (Yoshizawa et al., 1995). Calpain-1 requires 50-200 mM Ca2+ for half-maximal activity whereas calpain-2 requires significantly more (400-800 mM). Since both of these concentrations are well in excess of the typical levels of free intracellular Ca2+ (50-500 nM), exactly how the ubiquitous calpains are activated in vivo remains uncertain. It seems likely, however, that specific activating proteins are probably involved (Melloni et al., 1998). The ubiquitous calpains are also regulated by interaction with calpastatin, an endogenous inhibitor which has been identified in a diversity of cell types (Johnson & Guttmann, 1997).

Calpain-3 is the predominant calpain of adult mammalian skeletal muscle, its transcripts being expressed in this tissue at least 10x in excess of those of the ubiquitous calpains (Sorimachi et al., 1996). Calpain-3 does not form a complex with the small (30 kD) ubiquitous calpain subunit, but instead may exist as a homodimer (Kinbara et al., 1998). It also does not bind to calpastatin, the ubiquitous calpain inhibitor, but it may be stabilised in vivo through interaction with the large sarcomeric protein titin (connectin) in which there are two binding sites for calpain-3 (Kinbara et al., 1997). Since calpain-3 is active at sub-micromolar concentrations of intracellular Ca2+ it seems likely intermediate activating proteins are not required (Sorimachi, Ishiura & Suzuki, 1997).

Relatively little is known currently about calpain-6, the calpain identified only in embryonic skeletal muscle. However, mouse calpain-6 lacks the C-terminal calmodulin-like EF hand domain and the critical cysteine and histidine residues of the protease active site implying that it is not proteolytically active (Dear et al., 1997).

Mammalian calpains are differentially regulated during skeletal muscle development

The proteolytic activities of calpains -1 and -2 increase significantly during the differentiation of L6 rat myoblast-like cells in vitro, and their fusion to form myotubes can be inhibited by calpeptin, a cell-penetrating inhibitor of calpains (Ebisui et al., 1994). The regulation of ubiquitous calpain activity by calpastatin also changes during differentiation, with calpastatin protein levels diminishing markedly prior to myoblast fusion (Barnoy et al., 1999).

Calpain-2 first appears early in C2C12 mouse myoblast-like cells, whereas expression of calpain-1 occurs later during the final stages of muscle differentiation (Temm-Grove et al., 1999). As might be expected, calpain-2 antisense inhibits myoblast fusion (Balcerzak et al., 1998). Interestingly, expression of calpastatin in myoblast-like cultures occurs late in differentiation suggesting that the lack of this inhibitor may be crucial to the early stages of calpain-2 dependent fusion (Cottin et al., 1994). Indeed, microinjection of cultured myoblast-like cells with calpastatin inhibits their fusion (Temm-Grove et al., 1999).

Expression of calpain-3 increases during muscle differentiation (Poussard et al., 1996; Dickson & Evans, unpublished observations). Any possible role for calpain-6 in muscle differentiation is unknown, although it is unlikely to directly involve proteolytic cleavage since this isoform is not enzymatically active.

Calpain-3 exists as four splice variants in skeletal muscle

Calpain-3, the predominant calpain isoform in adult skeletal muscle, is subject to alternative splicing involving different combinations of the 24 exons in either the human or the mouse genomes (Richard et al., 1995; Richard & Beckmann, 1996). Splice removal of exon 6 (delta 6) impairs autolytic but not enzymatic (fodrinolytic) activity, whereas removal of exon 16 (delta 16) leads to increased binding to the titin and loss of enzymatic activity (Herasse et al., 1999). We have identified two other splice variants in muscle and other tissues of mice and humans in which exon 15 (delta 15) or exons 6, 15 and 16 (delta 6,15,16) are deleted (Dickson, Love and Evans, Genbank accession numbers AF 127765-6 ). Exon 15 encodes a consensus SV40-like nuclear localisation signal (NLS). Two other calpain-3 splice variants (Lp82 and Lp85) which have an alternatively spliced exon 1 are expressed only in the lens, possibly as a result of a lens specific promoter (Ma et al., 1998). The patterns of expression and the functions of calpain-3 and its splice variants during muscle differentiation are currently unclear. We hypothesise that calpain-3 and its splice variants are involved primarily in downstream events associated with muscle turnover and that they act to promote muscle fibre survival by mediating an anti-apoptotic effect.

Mutations in calpain-3 cause limb-girdle muscular dystrophy type 2A

Eight genetically distinct forms of human autosomally recessive limb-girdle muscular dystrophy (LGMD) are known currently, and genetic lesions underlying the development of seven of these have been identified to date (Moreira et al., 2000). Mutations in the gene coding for calpain-3 are responsible for one specific form of LGMD, namely LGMD-2A (Richard et al., 1995). At least 97 distinct pathogenic calpain 3 mutations distributed along the entire gene have been identified in LGMD-2A (Richard et al., 1999). Since not all of these mutations are in the active cysteine protease site or in the Ca2+-binding domain, exactly how their effects are manifested in LGMD-2A is unclear.

Significantly, all the calpain-3 mutants analysed in a study by Ono et al. (1998) showed loss of proteolytic activity implying functional conformational alteration in all of the different mutant proteins analysed. Changes in calpain-3 activity have not been correlated with other dystrophies, although up-regulation of calpains -1 and -2 has been reported in Duchenne and Becker progressive muscular dystrophies and in amyotrophic lateral sclerosis (Ueyama et al., 1998). It is thought that increased expression of calpain -1 and -2 isoforms in these diseases may be involved with myofibrillar degradation.

What is the role of calpain-3 in muscle development and disease?

The precise molecular functions of calpain-3 and its splice variants in the development of LGMD-2A remain to be fully determined. One strong possibility is that calpain-3 is involved in regulating apoptosis and that changes in expression of the different splice variants shift the dynamic balance between survival and death in muscle fibres. We hypothesise that muscle tissue wasting develops when fibre death exceeds the limited capacity of resident satellite cells to replenish the lost tissue. Thus in muscle diseases such as LGMD-2A, which is associated with mutations in the gene for calpain-3, the balance between fibre survival and death may be tipped in favour of apoptosis with wasting inevitably ensuing.

Our work on the calpains should enhance our understanding of the enigmatic nature of this family of enzymes and could provide a basis for the development novel treatment procedures for an unusual from of muscular dystrophy.

We have also initiated a study of the calpain family in the zebrafish, Danio rerio, which is a model system for studying many aspects of vertebrate development. A comprehensive library-based screen has highlighted a number of different calpains which are currently being identified through sequencing, and analysed for their patterns of expression in normal and mutant zebrafish forms. We are also using the zebrafish as a model organism to test the biological impact of environmental pollutants (Fraser et al., 2000; Palmer et al., 1998).

Research Project Availability

A number of different research projects focussing on calpains in mammalian and zebrafish experimental systems are available in the Evans laboratory. Please contact us if you wish to discuss things further and receive more specific details.

References

• Balcerzak, D, Cottin, P, Poussard, S, Cucuron, A, Brustis, JJ & Ducastaing, A (1998): Calpastatin-modulation of m-calpain activity is required for myoblast fusion. Eur J Cell Biol 75: 247-253.
• Barnoy, S, Zipser, Y, Glaser, T, Grimberg, Y & Kosower, NS (1999): Association of calpain (Ca2+-dependent thiol protease) with its endogenous inhibitor calpastatin in myoblasts. J Cell Biochem 74:522-531.
• Cottin, P, Brustis, JJ, Poussard, S, Elamrani, N, Broncard, S & Ducastaing, A (1994) Ca2+-dependent proteinases (calpains) and muscle cell differentiation. Biochim Biophys Acta 1223: 170-178.
• Dear, TN, Matena, K, Vingron, M & Boehm, T (1997): A new subfamily of vertebrate calpains lacking a calmodulin-like domain: implications for calpain regulation and evolution. Genomics 45: 175-184.
• Dear, TN & Boehm, T (1999): Diverse mRNA expression patterns of the mouse calpain genes Capn5, Capn6 and Capn11 during development. Mech Dev 89: 201-209.
• Ebisui, C, Tsujinaka, T, Kido, Y, Iijima, S, Yano, M, Shibata, H, Tanaka, T & Mori T (1994): Role of intracellular proteases in differentiation of L6 myoblast cells. Biochem Mol Biol Int 32: 515-521.
• Fraser, JK, Butler, CA, Timperley, MH and Evans, CW (2000): Formation of copper complexes in landfill leachate and their toxicity to zebrafish embryos. Environ Toxicol Chem 19: 1397-1402.
• Herasse, M & 11 others (1999): Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events. Mol Cell Biol 19: 4047-4055.
• Johnson, GV & Guttmann, RP (1997): Calpains: intact and active? BioEssays 19: 1011-1018.
• Kinbara, K, Sorimachi, H, Ishiura, H & Suzuki, K (1997): Muscle-specific calpain, p94, interacts with the extreme C-terminal region of connectin, a unique region flanked by two immunoglobulin C2 motifs. Arch Biochem Biophys 342: 99-107.
• Kinbara, K, Sorimachi, H, Ishiura, S & Suzuki, K (1998): Skeletal muscle-specific calpain, p94: structure and physiological function. Biochem Pharmacol 56: 415-420.
• Ma, H, Shih, M, Hata, I Fukiage, C, Azuma, M & Shearer, TR (1998): Protein for Lp82 calpain is expressed and enzymatically active in young rat lens. Exp Eye Res 67: 221-229.
• Melloni, E, Michetti, M, Salamino, F & Pontremoli, S (1998): Molecular and functional properties of a calpain activator protein specific for m-isoforms. J Biol Chem 273: 12827-12831.
• Moreira, ES & 10 others (2000): Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin. Nat Genet 24: 163-166.
• Ono , Y & 7 others (1998): Functional defects of a muscle-specific calpain, p94, caused by mutations associated with limb-girdle muscular dystrophy type 2A. J Biol Chem 273: 17073-17078.
• Palmer, FP, Butler, CA, Timperley, MH & Evans, CW (1998): Toxicity to embryo and adult zebrafish of copper complexes with two malonic acids as models for dissolved organic matter. Environ Toxicol Chem 17: 1538-1545
• Poussard, S & 6 others (1996): Evidence for implication of muscle-specific calpain (p94) in myofibrillar integrity. Cell Growth Differ 7: 1461-1469.
• Richard, I & 17 others (1995): Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 81: 27-40.
• Richard, I & Beckmann, JS (1996): Molecular cloning of mouse canp3, the gene associated with limb-girdle muscular dystrophy 2A in human. Mamm Genome 7: 377-9.
• Richard, I & 22 others (1999): Calpainopathy-a survey of mutations and polymorphisms. Am J Hum Genet 64: 1524-1540.
• Sorimachi, H & 7 others (1996): Highly conserved structure in the promoter region of the gene for muscle-specific calpain, p94. Biol Chem 377: 859-864.
• Sorimachi, H, Ishiura, S & Suzuki, K (1997): Structure and physiological function of calpains. Biochem J 328: 721-732.
• Temm-Grove, CJ, Wert, D, Thompson, VF, Allen, RE & Goll, DE (1999): Microinjection of calpastatin inhibits fusion in myoblasts. Exp Cell Res 247: 293-303.
• Ueyama, H, Kumamoto, T, Fujimoto, S, Murakami, T and Tsuda, T (1998): Expression of three calpain isoform genes in human skeletal muscles. J Neurol Sci 155: 163-169.
• Yoshizawa, T, Sorimachi, H, Tomioka, S, Ishiura, S & Suzuki, K (1995): A catalytic subunit of calpain possesses full proteolytic activity. FEBS Lett 358:101-103.

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Fish Antifreeze

2. Developmentally regulated expression of antifreeze in Antarctic fish

The Southern Ocean around the coast of Antarctica is a thermally stable environment in which the sea water temperature hovers close to its freezing point around -1.93C. Most marine teleosts have an equilibrium freezing point of about -0.7C, substantially higher than that of sea water, and they therefore cannot survive by supercooling in the icy Antarctic waters (DeVries et al., 1988). Notothenioid fishes thrive in this freezing environment, however, due to their capacity to produce antifreeze glycoproteins (AFGPs). AFGPs bind to and inhibit the growth of minute ice crystals that occasionally enter the fish, thus preventing their body fluids from freezing (DeVries and Cheng, 1992). They are a key evolutionary innovation that conferred freeze avoidance and empowered the adaptive radiation of the notothenioids in the poorly colonised, frigid waters of the Southern Ocean some 5-15 million years ago (Cheng et al., 1997; Eastman and McCune, 2000).

The site of AFGP synthesis in notothenioids is unknown

As many as 20 AFGP isoforms, all containing the same glycotripeptide, are synthesized in notothenioid species (Chen et al., 1997; Hsiao et al., 1990). The AFGPs evolved from a pancreatic trypsinogen-like protease (Chen et al., 1990; Cheng and Chen, 1990) and the isoforms are encoded by a large multigene family (Cheng, 1996). While the molecular basis for AFGP production is well understood, the site of AFGP synthesis remains an unresolved question. Unlike most other antifreeze-bearing fishes where the liver is the major synthetic site, adult notothenioids show only minimal AFGP transcription in this organ. Nothing is known about AFGP production in notothenioid larvae, nor when and where AFGPs are first synthesised during their embryogenesis.

In joint work with Art DeVries and Chris Cheng from the University of Illinois at Urbana-Champaign, we are currently investigating the sites of tissue synthesis of AFGPs in larval and adult dragonfish (Gymnodraco acuticeps), and the tempo of their upregulation during development. We are in an unique position to undertake these studies because we have ready access to larval, adult and fecund dragonfish in McMurdo Sound (Antarctica).

In preliminary work we have successfully fertilized mature oocytes in vitro, and maintained them throughout embryogenesis. We have also found that dragonfish eggs are laid in freezing seawater in early October. Although they are hypo-osmotic to seawater the eggs do not freeze, suggesting fortification with maternal antifreeze. Newly-hatched larvae swim directly to the surface-ice platelet layer to avoid predators, indicating that they contain AFGP and that a switch to zygotic production must take place during development.

We are using a combination of molecular biology (quantitative RT-PCR to enumerate transcript levels), immunocytochemistry (to localise the sites of AFGP expression and/or storage), and biochemistry (western blotting to identify specific AFGP isoforms) to analyse AFGP synthesis during development. Eggs at different times (before and after fertilisation) are being analysed in parallel for AFGP activity in the perivitelline, yolk and embryonic compartments using differential osmometry to estimate antifreeze activity.

Unique primers designed from dragonfish AFGP cDNAs will then be used in quantitative RT-PCR assays and specific AFGP antibodies will be employed for western blotting and immunocytochemistry in this combinatorial approach to determine the sites and tempo of AFGP synthesis during development.

Surviving the freezing waters of Antarctica demands tightly regulated control of AFGP production at all life stages because its synthesis requires a substantial energy investment. Our studies will open new pathways for gaining insight into how this control is achieved at the molecular and cellular levels. They will also generate information on the rates of larval development and growth relevant to Antarctic fisheries management in which New Zealand has increasing involvement.

Research Project Availability

A number of different research projects focussing on Antarctica are available in the Evans laboratory. Please contact Clive Evans if you wish to discuss the possibilities of Antarctic research further and receive more specific details.

References

• Chen, L, DeVries, AL and Cheng, C-HC (1997): Evolution of antifreeze glycoprotein gene from a trypsinogen gene in Antarctic notothenioid fish. Proc Natl Acad Sci 94: 3811-3816.
• Cheng, C-HC (1996): Genomic basis for antifreeze glycopeptide heterogeneity and abundance in Antarctic notothenioid fishes. In: Gene Expression and Manipulation in Aquatic Organisms, Soc. of Exp. Biol. Seminar Series 58 (Eds. S. Ennion & G. Goldspink), Cambridge University Press, pp. 1-20.
• Cheng, C-HC and Chen, L (1999): Evolution of an antifreeze glycoprotein. Nature 40: 443-444. DeVries, AL (1988): The role of antifreeze glycopeptides and peptides in the freezing avoidance of Antarctic fishes. Comp Biochem Physiol 90B: 611-621.
• DeVries, AL and Cheng, C-HC (1992): The role of antifreeze glycopeptides and peptides in the survival of cold water fishes. In: Water and Life: Comparative Analysis of Water Relationships at the Organismic, Cellular, and Molecular Levels (Eds. G.N. Somero, C.B. Osmond, C.L. Bolis), Springer Verlag, pp. 303-315.
• Eastman, JT and McCune, AR (2000): Fishes on the Antarctic continental shelf: evolution of a marine species flock?. J Fish Biol 57: 84-102.
• Hsiao, K-C, Cheng, C-HC, Fernandes, IE, Detrich, HW and DeVries, AL (1990): An antifreeze glycopeptide gene from the Antarctic cod Notothenia coriiceps neglecta encodes a polyprotein of high peptide copy number. Proc Natl Acad Sci 87: 9265-9269

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3. Molecular Ecotoxicology and Disease

There are a number of different research strategies available to assess biological impact at the molecular level although all have the same common goals: to provide a measure of the state of the environment though its impact on the biota and, where appropriate, to use the information obtained as a reference database for recommending any necessary environmental improvement. Our basic approach has been to use the extraordinary sensitivity of RT-PCR amplification as a tool in the quantitative assessment of the effects on fish of exposure to environmental pollutants and to use this as one measure of environmental impact (Evans et al., 2000, 2001; Miller et al., 1999).

Correlation between metallothionein induction as determined by quantitative competitive (qc) RT-PCR and hepatic copper levels in the yellowbelly flounder Rhombosolea leporina collected near Auckland, New Zealand.

In our initial studies this was achieved by measuring the induction of specific genes such as those for metallothionein (which reflects heavy metal exposure) and cytochrome P4501A (which reflects exposure to specific hydrocarbons). In order to assess the appropriateness of the RT-PCR approach, correlates have been sought between our molecular biological studies and levels of specific pollutants in fish tissues as well as with a spectrum of fish health indicators.

Stomach contents of Trematomus bernacchii collected near the sewage outfall at McMurdo Station, Antarctica. Peas, corn and carrot are visible in the opened stomach.

Molecular ecotoxicology offers a powerful approach which supplements the more classic procedures of environmental toxicology. Because it provides information at the earliest response level to exposure it has the capacity to serve as an early warning indicator of potentially more significant and widespread effects. Molecular ecotoxicology is thus a key parameter in monitoring the environment and recommending steps for any necessary improvement. Additionally, since knowledge of the molecular and cellular responses to particular pollutants also contributes to our understanding of the aetiology of specific bioeffects, the molecular ecotoxicology approach provides a route to the development of treatments for improving organismal health.

X-cell disease in the gills of Trematomus bernacchii collected from Winter Quarters Bay, Antarctica. The X cells are visible as large cells with distinct nucleoli in the basal region of the lamellae. Thrombus-like inclusions are obvious within aneurysms of the fused secondary gill lamaellae.

Research Project Availability

A number of different research projects focussing on Molecular Ecotoxicology are available in the Evans laboratory. Please contact us if you wish to receive more specific details.

References

• Evans, CW, Hills, JM and Dickson, JMJ (2000): Heavy metal pollution in Antarctica: a molecular ecotoxicological approach to exposure assessment. J Fish Biol 57A: 8-19.
• Evans, CW, Wilson, DA and Mills, GN (2001): Quantitative competitive RT-PCR as a tool in biomarker analysis. Biomarkers 6: 7-14.
• Miller, HC, Mills, GN, Bembo, DG, Macdonald, JA & Evans, CW (1999): Induction of cytochrome p450 1A (CYP1A) in Trematomus bernacchii as an indicator of environmental pollution in Antarctica: assessment by quantitative RT-PCR. Aquatic Toxicol 44: 183-193.

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Recent Publications