1. PLANTEAMIENTO METODOLOGICO
2.9. Residuos Solidos
In the preceding sections, the discovery of the mammalian enolases, and their expression and ontogeny have been discussed . A large body of evidence has established that the three subunits are encoded by three dispersed single-copy genes: this includes data from genomic Southern analysis, comparison o f cDNA sequences and the mapping of each cytogenetic locus to separate chromosomes in man, rat and mouse (Khan et al., 1974; Grzeschik, 1974; Van Cong et al., 1977; Cook and Hamerton, 1979; Law and Kao, 1982; Craig et al., 1989; Feo et al., 1990a; Mitchell et al., 1991; Goran Levan, pers. comm.); there is also a NNE pseudogene in the human genome (Feo et al., 1990b). When enolase isoenzyme heterogeneity in the rat was first discovered, the authors asked why different subunits, with indistinguishable kinetic properties, should coexist (Rider and Taylor, 1974; Fletcher et al., 1976). This question has remained pertinent, given the specific and independent regulation of the enolase genes as described above.
Introduction: Chapter I
In recent years, a large number of enolase sequences has been published (see Table 1.2) and this has facilitated comparisons between the three isoproteins to reveal differences in structure which may in turn provide some clues about their individual functional roles. The number of independently verified clones has virtually excluded
E n o la s e S p e c ie s S e q u e n c e R e f e r e n c e s C o m m e n t s
a (NNE) rat cDNA Sakimura el al., 1985a
mouse cDNA Kaghad et al., 1990
human cDNA
genomic cDNA
Giallongo et al., 1986
Giallongo et al., 1990
Verma and Kurl, 1990 Lung enolase (1)
duck cDNA
genomic
Wistow et al., 1988
Kim et al., 1991
a-enolase/t-crystallin (2)
chick cDNA Tanaka et al., 1995
P (MSE) rat cDNA
genomic Ohshima et al., 1989 Sakimura et al., 1990 mouse cDNA genomic Lamancte et al., 1989 Unpublished (3) human cDNA genomic Call et al., 1990 Peshavaria et al. ,1989
Peshavaria and Day, 1991
rabbit protein Chin, 1990
chick protein
cDNA
Russel et al., 1986
Tanaka et al., 1995
y (NSE) rat cDNA
cDNA genomic
Sakimura et al., 1985b
Forss-Petter et al., 1986
Sakimura et al., 1987
mouse cDNA Kaghad etal., 1990
human cDNA/protein cDNA cDNA cDNA genomic McAleese et al., 1988 Day etal., 1987
Van Obberghen et al., 1988;
Oliva etal., 1989
Oliva et al. ,1991
3' UTR only
Table 1.2: Origin of the known mammalian and avian enolase sequences. (1) Verma and Kurl (1990) have reported the sequence o f a cDNA encoding enolase which they isolated from a human lung
library. The lung enolase is most similar to NNE (Giallongo et al., 1986) but not identical; most
remarkably, the deduced amino acid sequence is 458 residues in length, 25 residues longer than all the other reported mammalian sequences. (2) The duck a-enolase gene encodes a bifunctional protein also
known as r-crystallin (see section 1.3). (3) The mouse MSE gene sequence (N. Lamandi, S. Brosset,
A. Keller, M. Lucas, and M. Lazar) is unpublished in the literature but is available from the databases under accession number X61600.
the problem of sequencing errors and it is therefore possible to compare the sequences of each subunit from mouse, rat and man without such errors confounding the observed variatioa Each polypeptide is 433 amino acid residues in length and perfect alignment between all nine sequences is possible; this allows both paralogous (within species between unlike subunits) and orthologous (across species between like subunits) comparisons to be made in all possible pairwise combinations (see Table
Introduction: Chapter I
1.3). It is also possible to align each mammalian sequence with that of S. cerevisiae enolase 1 (Holland et al., 1981), allowing any substitutions found between subunits to be assessed with respect to the secondary and tertiary structure predicted for the yeast protein (Lebidoa and Stec, 1988; Lebidoa et al., 1989; Stec and Lebidoa, 1990).
Species/ enolase
rNNE rNSE rMSE mNNE mNSE mMSE hNNE hNSE hMSE
rNNE 100 82.5 P 82.5 P 96 o . 82.5 83 82 84 rNSE 100 83 p 83 98,5., 84 84 i M S A ,
■
83.5 rMSE 100 82.5 81.5 o 82.5 82 97 o mNNE 100 83 p 84 p¡jgggg
83.5 83 mNSE 100 84 p 83■
82.5 mMSE 100 83 82 9 7 S o ' ■ hNNE 100 83 p 83 p hNSE 100 83.5 P hMSE 100Table 1.3: Comparison of the primary amino acid sequences of each enolase isoprotein deduced from rat (r), mouse (m) and human (h) cDNA clones. Figures represent percent identity over entire amino acid sequences excluding the initiator methionine (432 amino acids). Paralogous comparisons (p) are highlighted in light grey. Orthologous comparisons (o) are highlighted in dark grey. Where multiple
cDNA sequences exist, the following are used: human NSE - Oliva et al., 1990; rat NSE - Sakimura et
al., 1985a. The lung enolase isolated by Verma and Kurl (1990) is ignored in this table.
The results of such comparisons show that there is greater identity between like subunits across species, than there is between unlike subunits within species. This underscores biochemical and immunological evidence which has shown that, between species, like isoenzymes have similar pi values and like subunits are immunologically cross-reactive (Cardenas and Wold, 1971; Moore, 1975; Rider and Taylor, 1974;
1975a; Clark-Rosenberg and Marangos, 1980; Jackson et al., 1985; reviewed in Twyman and Jones, 1995c). Comparisons with the secondary structure predicted from crystallised yeast enolase showed that the major paralogous substitutions occurred at sites equivalent to yeast enolase 1 a-helices B, C, D, I and J which are presented on the surface of the protein and are not predicted to take part in substrate or cofactor binding (Day et al., 1993); the eight (3-strands which make up the active site o f the enzyme are invariant between the human subunits (Peshavaria et al., 1989). The kinetic similarity of the mammalian enolase isoenzymes is therefore thought to
Introduction: Chapter I
arise from the invariant core structure of the polypeptides, whilst functional
differences are thought to arise from surface properties which reflect interaction with other cellular components. Enolases throughout nature are well-known for secondary functions which are unrelated to catalysis: in the bacterium Clostridium difficile, enolase is thought to act as a toxin whilst in the yeast S. cerevisiae, enolase 1 is a heat shock protein (Green et al., 1993; Iida and Yahara, 1985); the role of a-enolase in certain birds and reptiles has already been discussed (Wistow et al., 1988). Compared to NNE, the expression of the tissue-specific enolases MSE and NSE is highly regulated and secondary functions are likely to reflect adaptations to specific intracellular environments. Hence, it has been shown that NSE is more resistant to chloride ion inactivation than NNE, and this might reflect an adaptation to the high intracellular chloride ion concentration of electrophysiologically active neurons (Marangos et al., 1978). Both NSE and MSE have been shown to be more thermotolerant than NNE although the physiological relevance of this is not clear (Marangos et al., 1978; Tanaka et al., 1985a). NSE has been shown to undergo slow component axonal transport and to be associated both with other enzymes and the membrane at the synaptic terminal (Brady and Lasek, 1981; Batke et al., 1988; Lim et al., 1983); such interactions would certainly require modifications to the surface structure of the protein. NSE has also been shown to act as a neuronal survival factor (Takei et al., 1991); these and other observations provide some suggestions as to why three enolase isogenes have evolved and have come to be expressed in the manner discussed above, however, there is still much to learn about the roles of these proteins. As well as providing data for functional autonomy amongst the enolases, sequence comparisons can provide some information about the evolution o f the enolase gene family. Paralogous comparisons in man, rat and mouse show that the divergence between isoproteins is approximately 17% in all pairwise combinations, suggesting that all three genes were created during a single evolutionary event (Day et al., 1993). The existence of an ancestral enolase gene is confirmed by the identical intron/exon architecture within the nine coding regions, with boundaries occurring at homologous positions in each sequence. Orthologous comparisons demonstrate that the burst event must have occurred before man/rodent speciation and the level of sequence identity observed between the limited bird enolase sequences and those of mammals indicate that it predated the divergence of birds and mammals (about 200 M yr ago); bird enolase genes also share the same intron/exon boundaries as their mammalian counterparts. Several authors have deduced that the event occurred approximately 300 M yr ago (Segil et al., 1984; Clark-Rosenberg and Marangos, 1980). This timescale would indicate that three enolases should exist in most tetrapods, although
Introduction: Chapter I
the specialisation seen in present day mammals and birds might not have arisen in all branches of the phylogenetic tree (see Twyman and Jones, 1995c). Orthologous comparisons also show how quickly each subunit is evolving: during man/rat speciation, only 8 amino acid substitutions occurred in the NSE subunit, 13 in the MSE subunit and 26 in the NNE subunit (Day et al., 1993); once again, the majority of these substitutions occurred on the surface of the protein. These data show that in mammals, NNE is evolving most quickly whilst the two tissue-specific subunits are evolving slowly, probably due to additional constraints on surface residues imposed by their probable secondary functions.
1.7 The molecular basis of enolase gene regulation