Aldehyde Dehydrogenase-derived O-Crystallins of Squid and Octopus SPECIALIZATION FOR LENS EXPRESSION*

Q-Crystallin of the octopus lens is related to aldehyde dehydrogenases (ALDH) of vertebrates (Tomarev, S. I., Zinovieva, R. D., and Piatigorsky, J. (1991) J. Biol. Chem. 266,24226-24231) and ALDHl/q-crystallin of elephant shrews (Wistow, G., and Kim, H. (1991) J. Mol. Euol. 32,262-269). Only very low amounts of Q-crystallin are present in the squid lens. Here, we have cloned Q-crystallin cDNAs of the octopus (Octopus dofleini) and squid (Ommastrephes sloani pacificus) lenses. The deduced amino acid sequences of Q-crystal-lin from these species are 78% identical to each other, 56-58% identical to cytoplasmic ALDHl and mito- chondrial ALDH2 of vertebrates (which are 66-68% identical to each other), and 40% identical to Esche- richia coli and spinach ALDHs. These data are consistent with the idea that the ALDHl/ALDH2 gene dupli- cation in vertebrates occurred after divergence of cephalopods from the line giving rise to vertebrates, but before the separation of squid and octopus. Southern blot hybridization indicated that Q-crystallin is en- coded by few genes (possibly just one) in octopus and squid. Northern blot hybridization revealed two bands (2.7 and 9.0 kilobases)

Q-Crystallin of the octopus lens is related to aldehyde dehydrogenases (ALDH) of vertebrates (Tomarev, S. I., Zinovieva 32,[262][263][264][265][266][267][268][269]. Only very low amounts of Qcrystallin are present in the squid lens. Here, we have cloned Q-crystallin cDNAs of the octopus (Octopus dofleini) and squid (Ommastrephes sloani pacificus) lenses. The deduced amino acid sequences of Q-crystallin from these species are 78% identical to each other, 56-58% identical to cytoplasmic ALDHl and mitochondrial ALDH2 of vertebrates (which are 66-68% identical to each other), and 40% identical to Escherichia coli and spinach ALDHs. These data are consistent with the idea that the ALDHl/ALDH2 gene duplication in vertebrates occurred after divergence of cephalopods from the line giving rise to vertebrates, but before the separation of squid and octopus. Southern blot hybridization indicated that Q-crystallin is encoded by few genes (possibly just one) in octopus and squid. Northern blot hybridization revealed two bands (2.7 and 9.0 kilobases) of Q-crystallin RNA in the octopus lens and one band (4.2 kilobases) in the squid lens; Q-crystallin RNAs were undetectable in numerous non-lens tissues of octopus and squid, suggesting lensspecific expression of this gene(s). Finally, extracts of the octopus lens had no detectable ALDH activity using different substrates, consistent with Q-crystallin having no enzymatic activity. Taken together, our results suggest that Q-crystallin evolved by duplication of an ancestral gene encoding ALDH and subsequently specialized for refraction in the transparent lens while losing ALDH activity and expression in other tissues.
The eyes of cephalopods (squid and octopus) and vertebrates show striking similarities, although they have been formed independently and are considered examples of convergent evolution (1). Despite their independent evolution, cephalopods and vertebrates have employed the same strategy of recruiting crystallins from pre-existing enzymes or stressrelated proteins. The major crystallins of squid and octopus, S-crystallins, are related (but not identical) to glutathione S-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted and L06903.
The octopus lens also has a less abundant crystallin, called Q-crystallin ( 7 ) , which is related to aldehyde dehydrogenase (ALDH)' (4). Since cytoplasmic ALDHl is the major crystallin (7-crystallin) in the mammalian elephant shrew (8), Qand 7-crystallins represent the first example of a similar enzyme-crystallin in vertebrates and invertebrates. An ALDH-related protein also comprises -70% of the watersoluble proteins in the lens of the light organ of the squid Euprymna scolopes (9), and the liver tumor-induced ALDH3 isoenzyme accumulates to high concentrations in the mammalian cornea, where it is suspected of having both structural and enzymatic roles (10)(11)(12)(13).
Only limited information is available on the structure of ALDH-related crystallins in cephalopods. The partial structures of five tryptic peptides covering -15% of the complete sequence of Q-crystallin in Octopus dofleini (4) and three peptides covering -9% of the sequence of the light organ lens protein in E. scolopes (9) have been determined. Determination of the complete structure of Q-crystallin would be very useful for elucidating its structural and functional relationships with ALDHs of vertebrates and with the major protein of the squid light organ lens. In this investigation, we have cloned Q-crystallin cDNAs of the squid and octopus lenses and showed that the sequences of their encoded proteins are equally similar to cytoplasmic ALDHl and mitochondrial ALDH2 of vertebrates.

MATERIALS AND METHODS
Obtaining Squid and Octopus-The octopus 0. dofleini and the squid 0. sloani pacificus were collected on the Russian Pacific coast (Vladivostok). The squid Ldigo opalescens was collected at the Hopkins Marine Station (Pacific Grove, CA). The lenses and specified organs were removed and stored in liquid nitrogen.
PCR of Octopus Q-Crystallin cDNA-Degenerate oligonucleotide

T)T(T/G)GA(T/C)AC(T/A)GA(A/G)CA(T/C)GG(T/A)CC(A/T) C A(G/A)AT(C/T)GA(T/C)GA(G/A)GA(G/A)CA(G/A)TA-3') and
oligonucleotide 6429 (5'-ACTGGACCGAAGATTTCTTCTTTA-GCGAT-3') corresponding to the tryptic peptides NT66 and NT104, respectively, of octopus Q-crystallin (4) were made on an automated DNA synthesizer (Model 380B, Applied Biosystems, Inc.) and used as primers for PCR using an octopus lens cDNA library (4) as template. Samples underwent 30 amplification cycles of denaturation at 94 "C for 2 min, followed by annealing at 50 "C for 2 min and extension at 72 "C for 3 min in an automated thermocycler (Perkin-Elmer Cetus Instruments). A fragment with the expected length of 200 bp was excised from an agarose gel, purified using Geneclean The abbreviations used are: ALDH, aldehyde dehydrogenase; PCR, polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s).

11449
(Bio 101, La Jolla, CA), and cloned in the Bluescript plasmid. Identity of the PCR fragment was confirmed by sequencing (see below). The PCR fragment was labeled with [32P]dCTP (3000 Ci/mmol; Amersham Corp.) using the random primer method (Bethesda Research Laboratories kit) to a specific activity near IO9 cpm/pg DNA.

Isolation and Sequencing of cDNA Clones Encoding Squid and
Octopus Q-Crystallins-About lo3 plaques of an octopus (4) or 5 X 10' plaques of a squid (5) lens cDNA library were screened with the 32P-labeled 200-bp PCR fragment of Q-crystallin cDNA (see above; positions 1059-1282 in Fig. 1) or with the 1000-bp PCR fragment (positions 220-1176 in Fig. 1) generated after isolation and characterization of the complete octopus Q-crystallin cDNA. Conditions for screening were as described (4), with the exception that hybridization with the squid library was conducted at 37 "C and final washings were at 50 "C. The recombinant bacteriophages hybridizing with Qcrystallin-specific probes were converted to Bluescript SK(-) plasmids according to the manufacturer's instructions, and the plasmid DNA was isolated using Qiagen columns (Qiagen Inc., Studio City, CA). The cDNA inserts were sequenced by the dideoxynucleotide termination method (14) using Sequenase Version 2.0 (U. S. Biochemical Corp.) and 3SS-dATP (1000 Ci/mmol; Amersham Corp.). Synthetic oligonucleotides were used as sequencing primers. Sequences were determined for both strands of DNA. DNA sequences were analyzed using the GCG program package (15). The evolutionary relationships of cephalopod Q-crystallins and different ALDHs were calculated using the PROTPARS program of the Phylip package (16) and the DISTANCES program of the GCG package (15).
Northern and Southern Blot Analyses-RNA was isolated from different organs of squid and octopus by the acidic guanidinium thiocyanate/phenol/chloroform extraction method (RNazol B, Cinna/Biotech, Friendswood, TX) (17) and stored at -70 "C. Total RNAs were size-fractionated by electrophoresis on 1.5% agarose, 2.2 M formaldehyde gels; transferred to Nitran nylon membrane filters as recommended by the manufacturer (Schleicher & Schuell); crosslinked by ultraviolet irradiation (Stratalinker, Stratagene); and hybridized with different 3ZP-labeled probes as described (4). High molecular weight squid and octopus DNAs were isolated from ovaries or testes (18), digested with different restriction enzymes, separated by electrophoresis on 0.8% agarose gels, transferred to Nitran nylon filters, cross-linked by ultraviolet radiation, and hybridized with 32Plabeled probes as described (5).

Isolation and Characterization of cDNA Clones Encoding Squid and
Octopus Q-Crystallins-One positive plaque (pOAL1) was identified among lo3 plaques of an octopus lens cDNA library after screening with an Q-crystallin-specific PCR probe obtained as described under "Materials and Methods." pOAL1 cDNA ( Fig. 1) contains an open reading frame encoding a protein of 495 residues excluding the first methionine (see Fig. 2). The sequences of all five tryptic peptides determined earlier (4) were identified within the deduced amino acid sequence of pOAL1. Two differences were found: position 1 of peptide NT79(2) has an isoleucine instead of an alanine, and position 9 of peptide NT83 has an isoleucine replacing phenylalanine (see Fig. 2). These differences may represent polymorphisms that appear common among natural populations of cephalopods (5, 6). The 5"untranslated sequence of pOALl has a length of 83 bp, and the 3"untranslated sequence has a length of 225 bp. An AATAAA polyadenylation signal is found 34 bp upstream of the poly(A) tail, which is slightly further upstream than typically found among mRNAs.
We next isolated Q-crystallin cDNA from the squid 0. sloani pacificus. Three plaques giving hybridization signals with the octopus Q-crystallin probe were isolated as described under "Materials and Methods." The combined nucleotide sequence of the two longest overlapping cDNAs (pSAL) is shown in Fig. 1. It is much longer (3831 bp) than the longest octopus Q-crystallin cDNA (1724 bp), but encodes a protein that is only 1 amino acid shorter (494 residues) and clearly homologous to the octopus Q-crystallin (see below). The 5'-and 3'untranslated regions are both longer in the squid Q-crystallin cDNA (167 nucleotides and 2176 nucleotides, respectively) than in the octopus cDNA. The squid polyadenylation signal is located 16 nucleotides upstream of the poly(A) tract. The octopus and squid Q-crystallin mRNAs are 74% identical in the coding region; there are no significant regions of similarity in the untranslated regions.
Assays for ALDH Actiuity-In view of the structural relationship of 0-crystallin with ALDH of vertebrates, extracts of the octopus lens were assayed for ALDH activity; extracts from the octopus digestive gland were also examined for comparison. Three concentrations were examined for each substrate tested (see "Materials and Methods"). The digestive gland extracts gave activity with all substrates tested. The maximum activity was observed with 1 and 10 mM acetaldehyde and propionaldehyde or with 200 p M hexanal and OCtanal. The digestive gland ALDH activity ranged from 0.008 to 0.019 units/mg of protein with different substrates and in different experiments. The lens extracts had virtually no detectable ALDH activity with any of the substrates tested. Since Q-crystallin is clearly more abundant in the lens than are proteins of similar molecular mass in the digestive gland (Fig. 3), these results indicate that 9-crystallin possesses no ALDH activity with these commonly used substrates. As in these tests on ALDH activity in the eye lens, the light organ lens of E. scolopes, which also has an ALDH-like protein as its major crystallin, did not show significant ALDH activity (9). The same was true for 11-crystallin of the elephant shrew, which also had no ALDH activity (8).
Lens-specific Expression of Squid and Octopus Q-Crystallins-we next examined the tissue specificity of squid and octopus Q-crystallin gene expression by Northern blot hybrid- TT TAC TAGTTCACCATTTTTTCTATATATTTTACATTAGCTTGGCTTT~TATTGCCAMTGTAAAMTCTGCAAACGAMTATTCTATCATTGAATTAGTAGATTCATGATTCTTAGAG 2372

T C T T C T T C A T C A T T C C T G C M T G A T T T C G G A G C C M G~T M A C G A C A T T T T A A T A C M T A T A T T C A T T A A A A T T A T C A A T~A T C A M T T T T A T A A A~T G M T A A A T C A G A A 2732
AGGTAAAMCATCCGCCATTATTTTTGGCGATTAATGATTTCCTCTTCGTCGATGATATTTGMGACGATAAMGCMCATCMGCGATATTGTGATTCATTTATCTMATMAATAACT 2 8 5 2

2972
CCCAGTGTTTATGMTCTCATATGACTGAAAGACTAAGAAAAGGAACATCTMTAATATGTTCCATAAACTTTTCATTCTTCAGTTTTAATTTTAAGATATTTTTTACAGTMGTTTATT 3092 ization (Fig. 4A). Labeled probes corresponding to different parts of Q-crystallin cDNA (positions 1-250, 220-1176, and 1404-1674) were used in these experiments and gave identical results. They hybridized exclusively with lens RNA; and in all cases, two hybridization bands of -2650 and 9000 nucleotides in length were observed (Fig. 44). Since there is probably only one copy of the Q-crystallin gene in the octopus genome (see below), these results may reflect the usage of two polyadenylation signals, although this requires further investigation. The squid 0. sloani pacificus Q-crystallin cDNA also hybridized exclusively with both homologous and heterologous lens RNAs (Fig. 4B). In contrast to the two bands of Qcrystallin mRNA obtained in the octopus lens, only one hybridization band of -4200 nucleotides in length was observed in the squid lenses. These results suggest that the squid and octopus Q-crystallin mRNAs are lens-specific.   ALDHl (19) and ALDHS (20) and betaine ALDH of E. coli (ALDHB) (23). Sequenced peptides of ALDH-like crystallin from the E. 294-2646 of the octopus and squid cDNAs, respectively, were used as probes. With the octopus, two 0-crystallin hybridization bands of -1.2 and 4.45 kbp were present in the EcoRIdigested DNA, and two bands of -1.9 and 4.6 kbp were present in the HindIII-digested DNA (Fig. 5A). The hybridization patterns of the squid DNA varied with the restriction enzyme. Five bands (1.0, 1.45, 1.8, 3.9, and 6.2 kbp) were obtained after digestion with EcoRI; two bands (2.2 and 11 kbp) were evident after digestion with HindIII; three bands (13,15, and 20 kbp) were present after digestion with BamHI; and only a single band (10.5 kbp) was seen after digestion with SalI (Fig.   5B). Multiplicity of hybridization bands in some restriction digests can be partially explained by polymorphism, which appears common in natural squid populations (5,6). Although further experiments are necessary, our data are consistent with the existence of few genes (and perhaps only one) for Qcrystallin in these species.

DISCUSSION
The ALDH-related Q-crystallin of cephalopods (4) and ALDHl/q-crystallin of elephant shrews (8) are the only known examples of similar lens crystallins in invertebrates and vertebrates. They show that independent choices during the convergent evolution of lenses in cephalopods and mammals have resulted in a similar protein for a crystallin. Our earlier protein gel data (4) as well as recent Western blot experiments (9) indicate that the octopus lens has much more Q-crystallin compared with the squid lens. The hybridization data presented here establish that Q-crystallin is present in the squid lens, although at a concentration generally considered too low to qualify as a crystallin. Surveys among vertebrates lenses have also shown that taxon-specific crystallins that exist in abundance in one species may be present at low concentrations in other related species (8). In the squid E. scolopes, ALDH-like crystallins are the major proteins of the light organ lens (9). In preliminary experiments during the course of this study, we partially sequenced a PCR-generated 650-bp cDNA fragment of Q-crystallin of the eye lens of E.
scolopes (data not shown). The sequences of two peptides (V28 and T44) of the ALDH-like protein from the light organ lens (9) were identified in the deduced amino acid sequence of the ocular R-crystallin of E. scolopes (see Fig. 2). Indeed, the available sequences of ocular R-crystallin and light organ Lcrystallin suggest that both proteins are identical.2 Since the cephalopod eye lens is derived from two ectodermal layers formed on the dorsal surface of the head lobe (33) and the light organ lens is derived from muscle of the hindgut (34), we conclude that the ALDH-related R-crystallin has been independently recruited to serve similar structural functions even in different tissues of the same species. A diagram of the relationships between R-crystallins and different ALDHs is shown in Fig. 6. In view of our finding that R-crystallins of squid and octopus are 78% identical to each other and 5648% identical to cytoplasmic ALDHl and mitochondrial ALDH2 of mammals, one may speculate that the gene duplication giving rise to ALDHl and ALDH2 (which are 66-68% identical) occurred after the divergence of cephalopods from the line leading to mammals (estimated at * R. D. Zinovieva, S. I. Tomarev 200-300 million years (35)). This speculation does not consider, of course, possible differences in the rates of evolution of R-crystallins in the lens and enzymatically active ALDHs. It is interesting to note, however, that R-crystallins and ALDHl/ALDH2 are each -40% identical to E. coli (23) and spinach (24) ALDHs (see Fig. 6), consistent with the possibility that R-crystallins and enzymatic ALDHs have evolved at similar rates. In contrast to the relatively high conservation between R-crystallins of cephalopods and ALDHs of other species, the glutathione Stransferase-related S-crystallins of squid and octopus lenses show only -25% identity to glutathione S-transferases of vertebrates (3-5) and as little as 42-44% identity to glutathidata.

R-Crystallin/Aldehyde
one S-transferase of squid (6). This suggests that glutathione respectively. ALDH Plant and ALDH E. coli refer to betaine ALDHs of spinach (24) and E. coli (23), respectively; Q-Cr Oc and Q-Cr Sq refer to octopus and squid Q-crystallins, respectively. The length of the branches is proportional to the relative phylogenetic distance between the proteins, which was calculated using the DISTANCES program (15). The standard deviations are shown for several branch points where more than two sequences were compared.
S-transferases and S-crystallins have evolved faster than ALDHs and Q-crystallins.
One of the characteristic features of enzyme-crystallins of vertebrates is that the identical gene encodes the enzyme and the crystallin (36). Thus, vertebrate enzyme-crystallins are generally expressed at low concentrations in non-lens tissues when performing their metabolic functions and at high concentrations in the lens when fulfilling structural roles. In some cases, the enzyme-crystallin gene has duplicated, such as with &crystallin, and one of the duplicated genes has specialized for lens expression. Even when this happens, however, both genes are still expressed in non-lens tissues (37, 38), and the gene encoding the enzyme may still be expressed abundantly in the lens, where it probably serves a refractive function (39). In contrast to the situation among vertebrate enzyme-crystallins, our recent studies on S-crystallins of squid showed that the glutathione S-transferase gene from which the S-crystallin family was derived is barely expressed in the lens and that the S-crystallins are found only in the refractive lens and cornea (4)(5)(6)13). The present study suggests that a similar separation of function has occurred with ALDH/Q-crystallin in cephalopods and that Q-crystallin and ALDH are encoded in different genes. This is consistent with the fact that ALDH activity is much higher in the digestive gland than in the lens, where activity was not detectable, although we cannot eliminate the possibilities that &crystallin possesses enzymatic activity with substrates not tested in this study, that an ALDH inhibitor exists in the lens extracts, or that ALDH activity is suppressed by post-translational modification(s) of Q-crystallin in the lens. Replacement of Cys-302, which is located at the active site of ALDHs (26,27, 29,30), by Arg in both squid and octopus Q-crystallins also argues that Q-crystallins do not possess ALDH activity. The Northern blot experiments showed that Q-crystallin mRNA is abundant in the lens and undetectable in other tissues, including the digestive glands of octopus and squid, which also supports the idea that genes with different expression patterns encode Q-crystallin and ALDH. In contrast to the -10 genes encoding the S-crystallins (5), Q-crystallin appears to be a much smaller family, possibly consisting of a single member. Further experiments are necessary to establish the number of Q-crystallin genes and to characterize the ALDH genes of cephalopods. We anticipate that site-directed mutagenesis of Q-crystallin cDNA and determination of the structure of active ALDHs from squid and octopus will help clarify the role of different residues in enzymatic activity.
Finally, the independent recruitment of ALDH-like proteins to be crystallins in the eye lenses of cephalopods (4) and elephant shrews (8) and in the light organ lens of squid (9) is consistent with the generality that crystallins are identical or related to proteins involved in stress responses (40, 41). Protection against oxidative stress and protein denaturation have been considered as factors in the recruitment of crystallins, especially in view of the deleterious effects for transparency of agents leading to protein denaturation and aggregation (42). Indeed, in vertebrates, a-crystallins protect the lens by functioning as a molecular chaperone (43), and many of the taxon-specific crystallins are related or identical to detoxification enzymes (36, 41). Protection against oxidative stress may be especially important for cephalopod lenses since seawater contains high concentrations of photochemically generated oxygen radicals and redox-active transition metal ions (see Ref. 44). Moreover, the squid light organ contains a high concentration of peroxidases3 that may generate many potentially dangerous oxidants, such as superoxide anion, hydroxyl free radicals, and hypochlorous acid (45, 46), threatening the lens. Thus, the recruitment of Q-crystallins may have initially involved induction of enzyme activity for stress protection and subsequent gene duplication and specialization for refraction in the eye and light organ.