Characterization of γS-Crystallin Isoforms from Lip Shark (Chiloscyllium colax): Evolutionary Comparison between γS and β/γ Crystallins

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS240, 51 – 56 (1997)

ARTICLE NO.RC977600

Characterization of

g

S-Crystallin Isoforms from Lip Shark

(Chiloscyllium colax): Evolutionary Comparison

between

g

S and

b

/

g

Crystallins

1

Fu-Ming Pan,* Ming-Hong Chuang,† and Shyh-Horng Chiou*

,

_†

,2

Laboratory of Crystallin Research, †Institute of Biochemical Sciences, National Taiwan University,

and *Institute of Biological Chemistry, Academia, P.O. Box 23-106, Taipei, Taiwan

Received September 30, 1997

The abundant presence of various common and

spe-gS-Crystallin from shark eye lenses, formerly termed

_{cific classes of structurally conserved proteins in eye}

bs crystallin in mammalian lenses, is structurally char-

_{lenses (crystallins) of different species of vertebrates}

acterized in this study by cDNA cloning and sequencing.

_{constitutes a good model system to unravel the complex}

To facilitate sequence characterization of gS-crystallin

_{process of evolution in structurally homologous}

pro-possessing intermediate structural properties between

_{teins (1,2). Fish represents the oldest and most diverse}

b- and g-crystallins, cDNA mixture was constructed

_{group of vertebrates. The modern fishes comprise two}

from the poly(A)

/

_{mRNA isolated from shark eye lenses,}

major classes of piscine species: [1] Osteichthyes or

te-and amplification by polymerase chain reaction (PCR)

_{leostean (bony) fishes and [2] Chondrichthyes or}

carti-was carried out to obtain nucleotide segments encoding

_{laginous fishes (sharks and skates).}

multiple shark gS-crystallins. Sequencing several posi-

_{The study of lens crystallins from the lowest piscine}

tive clones revealed that a multiplicity of isoforms exists

_{class is of special interest from the evolutionary point}

in the gS-crystallin class of this cartilaginous fish,

simi-of view because they constitute the early protein forms

lar to authentic g-crystallin family characterized from

of vertebrates and are thought to have been ancestral

the same shark species. Comparison of protein

se-to those of land vertebrates. Sharks diverged from the

quences encoded by two representative shark gS1 and

Placodermi (one class of armored fishes) long before the

gS2 cDNAs with those published sequences of b-, g-, and

appearance of modern bony fishes and amphibians (3).

gS crystallins from bovine, human, bullfrog and carp

The characterization of shark crystallins would be

lenses indicated that there is about 35–64% sequence

deemed very important for the phylogenetic

compari-homology between shark gS crystallins and structurally

son in light of the recent elucidation of the complete

related crystallins from different evolutionary classes,

sequences of

g

-crystallins from several species of

tele-with a higher sequence similarity between shark gS and

ostean fishes in Osteichthyes (4 – 6).

mammalian g-crystallins than that of shark gS and carp

The present study was performed in the endeavor to

gS or bovine gS crystallins. A phylogenetic tree

con-have a structural characterization of one unique class

structed on the basis of the sequence divergence among

of lens crystallins with their primary structure lying

various b-, g-, and gS crystallins corroborates the closer

between the well-known

b

- and

g

-crystallins. This class

relatedness of shark gS to authentic g-crystallin than

of crystallin, formerly called

b

S and now renamed

g

S

to mammalian and teleostean gS crystallins. It further

crystallin (7,8), exists as a monomeric protein which is

strengthens the supposition that ancestral precursors

similar to the major authentic

g

-crystallins. However

of gS-crystallins were present in the shark lens long

before the appearance of present-day teleostean and

unlike

g

-crystallins which possess a free N-terminal

mammalian gS-crystallins.

q 1997 Academic Press

amino-acid residue,

g

S-crystallin has a blocked N

ter-minus as most members of

b

-crystallin family. In this

report we have for the first time cloned and sequenced

g

S crystallins from the shark eye lenses, which possess

some structural properties distinguishable from those

1_{The sequence data of cDNAs for sharkgS-crystallins have been}

deposited in the EMBL Data Library under the Accession Numbers

_{of the existing}

_g

_{S-crystallins characterized from}

mam-X79226 and X79227 forgS1 andgS2, respectively.

malian species. The results indicate that

g

S crystallin

2_{Corresponding address: Institute of Biochemical Sciences,}

Na-characterized from the lower class of cartilaginous fish

tional Taiwan University, P.O. Box 23-106, Taipei, Taiwan. Fax:

Vol. 240, No. 1, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. Nucleotide and deduced protein sequences of sharkgS-1 (A) and sharkgS-2 (B) crystallins. In (A) the nucleotide sequence of 522-base pairs is shown above the amino-acid sequence of 173 residues including translation initiation methionine; in (B) the nucleotide sequence comprises 524-base pairs encoding a protein sequence of 173 amino acids. Asterisks (*) are indicated in every 10-nucleotide segment for easy tracing of sequence contents. Amino acids are denoted by one-letter symbols. The 5* and 3* nucleotide segments used as primers for PCR reactions are underlined.

cDNA amplification by PCR, cloning and sequencing ofg

S-crys-mammalian class, consists of a multitude of isoforms,

tallin isoforms. Two oligonucleotide primers of sense and antisense

and are more related to

g

-crystallin than

g

S or

b

-crys-orientations, covering 5*- and 3*-nucleotide coding regions forN- and

tallins based on sequence alignment and phylogenetic

C-terminal 4-6 amino-acid segments of the previously determined

comparison.

_{cDNA sequence for one carp gS-crystallin (10), with the forward}

sequence, 5_{*-CATGGGCAAG(A/G)TCA(T/C)CTT(C/T)-3* (19-mer)} and the reverse sequence, 5*-C(A/G)TCACTCCA(T/C)(G/A)A(T/

MATERIALS AND METHODS

G)GCG-3_{* (17-mer) (with slant lines indicating use of degenerate} codons in the primers) were synthesized. The conditions for PCR

Isolation of mRNA from shark lenses. The small shark

(Chiloscyl-reactions were similar to the previous reports for cDNA amplification

lium colax, brownbanded bambooshark or brown-spotted catshark

of teleostean lenses (5,6), i.e. subjecting to 40 cycles of heat denatur-as commonly called) of less than 1-year-old wdenatur-as obtained from a local

ation at 94_{7C for 2.5 min, annealing the primers to the DNAs at 487C} aquarium shop under a special contract for scientific research. Shark

for 1 min and 20 sec and running DNA chain extension with Taq lenses were removed and stored in liquid-nitrogen container

immedi-polymerase at 72_{7C for 3 min, followed by a final extension at 72 7C} ately after they were dissected and before the processing for mRNA

for 10 min. Products were treated with Klenow Fragment and T4 isolation. Two deep-frozen lenses from one shark were homogenized

polynucleotide kinase, and separated on a 1.2 % agarose gel and and RNA was extracted according to the standard cloning manual

electroeluted according to standard procedures. The DNA fragments of Maniatis et al. (9). To obtain a full-length crystallin cDNA,

were subcloned into pUC18 previously digested with SmaI/BAP, and poly(A)/_{RNA was purified using QuickPrep mRNA preparation kit}

then transformed into E. coli strain JM 109. Plasmids purified from (Pharmacia, Uppsala, Sweden) and then subjected to the synthesis

positive clones were prepared for nucleotide sequencing by dideoxy-of cDNA mixture by cDNA Synthesis System/Plus kit (Amersham,

deter-Vol. 240, No. 1, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1—Continued

mined by conventional isotope-labeled manual method was double- align highly evolved gene families that have clear evolutionary rela-checked by automatic fluorescence-based sequencing of templates tionships.

amplified by PCR using model 373A DNA sequencing System (Ap-plied Biosystems Inc., CA, USA) with a Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems Inc.).

RESULTS AND DISCUSSION

Sequence comparison of deduced sharkgS-crystallins and homol-ogy search. A commercially available software package (DNASTAR Inc., Madison, WI, U.S.A.) was used for the estimation of sequence

There were sharks in the oceans of earth long before

homology based on percent sequence identity (5).

the first animals had begun to colonize the land

sur-Construction of a phylogenetic tree forb-,g- andgS-crystallins of

face. Their history stretches back for at least 700

mil-vertebrate species. A software package of LaserGene for the Apple

lion years, a vast period of time as compared with

Macintosh computer from DNASTAR, Inc. was used for the

estima-shorter evolutionary history of other vertebrates (3).

tion of sequence homology based on percent similarity and divergence

among different cDNA sequences ofb-,g- andgS-crystallins. Percent

_{That sharks were once considered to be the most}

primi-divergence is calculated by comparing sequence pairs in relation to

_{tive are now thought to have been relatively specialized}

the phylogenetic tree. On the other hand the percent similarity is

regarding their complex biology to be ranked with birds

estimated by comparing sequences directly without accounting for

and mammals as highly evolved creatures. In this

re-phylogenetic relationships. Phylogenetic tree was then constructed

using the algorithm of Hein (12) in the MegAlign programs of the

port the structural characterization of

g

S-crystallins

package. It is a multiple-sequence alignment program that builds

_{from shark lenses by PCR and its comparison with}

trees as it aligns DNA or protein sequences using a combination of

those of teleostean and mammalian lenses is of special

distance matrix and approximate parsimony methods. This method

important to unravel the complex evolutionary history

constructs multiple-alignment by imposing restrictions based on

Vol. 240, No. 1, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 2. Multiple sequence alignment and comparison of seven crystallin sequences from species of different classes. The identical amino-acid residues among various sequences based on the first one (sharkgS-1) were expressed in white letters against black-background blocks. The gaps were introduced for optimal alignment and maximum homology for the sequences. Note that the middle region (residues# 70 – 130) shows a greater sequence variation than the N- and C-terminal regions among the compared sequences. Amino acid residues are denoted by one-letter symbols.

some conformational properties as revealed by circular

Characterization of cDNAs Encoding

g

S-Crystallins

dichroism (14,15). Shark

g

-crystallin showed a much

from Shark Lenses

more complex pattern in isoelectric focusing (data not

Previous studies have indicated the unusual struc-

_{shown), revealing that it consists of various charge}

iso-tural characteristics of shark

g

-crystallin as compared

_{forms (13, 14). Due to the complexity of}

_g

_-crystallin,

with those associated with the lenses of teleostean

_{we suspect that the same multiplicity of isoforms may}

fishes such as carp (13 – 15). Especially noteworthy is

_{be also present for}

_g

_{S-crystallin, a lens protein with}

the finding that the amino acid compositions of

g

-crys-

_{dual structural properties of}

_b

_{- and}

_g

_{-crystallins (7,8}

tallins seem to lack the unique characteristic of high

_{and the references therein). We have hence resorted to}

methionine content (ú 10%) as commonly observed for

_{the recent rapid method of cloning and sequencing by}

that of teleostean fishes (5,6). In contrast it is closer to

_{means of PCR technique for the determination of cDNA}

mammalian

g

-crystallins regarding both the amino

_{sequences of these multiple isoforms.}

acid composition,

N-terminal partial sequence plus

_{PCR amplification of total lens cDNA mixtures}

pre-pared from two lenses of a single shark with the

de-signed primers based on partial DNA coding sequences

of carp

g

S-crystallin (10) achieved the isolation of one

PCR fragment corresponding to the complete reading

frame encoding at least two

g

S-crystallin isoforms from

this shark species. The size determination of

PCR-am-FIG. 3. Pair-wise comparison of amino-acid sequence homology

plified cDNA coding for

g

S crystallin was estimated to

between two sharkgS-crystallins and various b-, g- andgS-crys-

_{be about 520 bp, in agreement with a protein of about}

tallins from species of different classes. Analysis of sequence homol-

_{170 – 180 amino-acid residues for mammalian}

_g

_{- and}

ogy was carried out using the software package (DNASTAR Inc.,

g

S-crystallins. The PCR-amplified DNA fragments

Madison, WI, USA) on the published sequences of carpgS (10),

bo-were then subcloned into pUC18 previously digested

vine gS (7), bullfrogb2 (24), bovine b2 (25), bovine gII (26) and

Vol. 240, No. 1, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 4. Pair-wise comparison of protein sequence similarity and divergence (Top) and construction of phylogenetic tree (Bottom) of eight crystallin sequences from various species of three major classes of vertebrates. Analysis of sequence data was carried out in a software package of LaserGene for the Apple Macintosh computer (DNASTAR Inc., Madison, WI, U.S.A.). Percent divergence is calculated by comparing sequence pairs in relation to the relative positions in the phylogenetic tree. On the other hand the percent similarity is estimated by comparing percent sequence identity directly without accounting for phylogenetic relationships. A phylogenetic tree was then constructed based on the percent divergence between protein sequences using a combination of distance matrix and approximate parsimony methods in the phylogeny generation program of Hein (12). This algorithm carries out multiple-alignment by imposing restrictions based on evolutionary relatedness of the aligned sequence pairs. The tree was built using clustal method and weighted residue-weight table. The length of each pair of branches represents the sequence distance between aligned pairs. The scale beneath the tree measures the distance between sequences (in millions of years). Bovine and bullfrogbB2 crystallins are included to indicate the fact thatb- andg-crystallins form ab/g

superfamily.

strain JM 109. Plasmids purified from two positive

173 amino-acid residues including the initiating

methi-onine, which is close to carp

g

S (174 a.a.) and slightly

clones were then prepared for nucleotide sequencing.

lower than bovine

g

S (178 a.a.).

Sequence Analysis of cDNA Encoding Shark

Sequence Alignment and Comparison of

b

-,

g

- and

g

S-Crystallins

g

S-Crystallins from Different Species

More than five positive clones have been identified,

with their 5

* and 3* nucleotide sequences being deter-

Fig. 2 aligns seven sequences encompassing

repre-sentative

b

-,

g

- and

g

S-crystallins from characterized

mined to be essentially identical to those of the

de-signed primers, indicative of the existence of multiple

crystallins of the major classes in vertebrates, which

have all been deduced from nucleotide sequences

cod-isoforms for shark

g

S-crystallin, which is in contrast

to bovine (7,8) and human

g

S-crystallins with only one

ing for these crystallins. It is noteworthy that there is

only about 35 – 64% sequence homology between shark

sequence being identified (16). In order to avoid

se-quencing errors introduced in the manual sese-quencing

g

S crystallins and structurally related

b

-,

g

- and

g

S-crystallins from different evolutionary classes.

How-of isotope-labeled cDNA, sequence accuracy was doubly

checked and confirmed by automatic fluorescence-

ever one salient feature is that some of the key residues

(such as Tyr-6, Glu-7, Phe-11, Gly-13 and Ser-34 based

based sequencing technique. The only uncertainty may

lie in the first and last few nucleotides present in the

on bovine

g

II sequence numbering) for the

mainte-nance of stability in

g

-crystallins (17 – 19) are mostly

5

* and 3* region of the PCR fragment even though we

have used some degenerate codons in the primers. Fur-

retained and conserved in all

b

-,

g

- and

g

S-sequences

even from species of distantly related classes, attesting

ther genomic analysis of shark

g

S-crystallins my help

solve the ambiguity in these short segments. The de-

to the conservative structural aspects of

b

/

g

superfam-ily. It is also of interest to find that N- and C-terminal

duced protein sequences together with their genetic

coding sequences of two clones, designated as shark

regions of these crystallins are more conserved than the

middle regions of the sequences (residues# 70 – 130).

g

S-1 and

g

S-2 are shown in Fig. 1A and 1B. The cDNA

sequences encoding shark

g

S-1 and

g

S-2 were both

In the pair-wise sequence homology comparison (Fig.

3) of various deduced amino-acid sequences from

spe-found to consist of 519 nucleotides excluding the stop

Vol. 240, No. 1, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

package (DNASTAR program), it is found that shark

REFERENCES

g

S-crystallins show 62 – 64% sequence identity to bo-

_{1. Chiou, S.-H. (1986) FEBS Lett. 201, 69 – 73.}

vine

g

II crystallins whereas only 52 – 56% sequence

_{2. de Jong, W. W., and Hendriks, W. (1986) J. Mol. Evol. 24, 121 –}

identity is found between shark and bovine

g

S-crys-

129.

3. Keeton, W. T. (1972) in Biological Science, 2nd ed., W. W.

Nor-tallins, indicating that shark

g

S is evolutionarily more

ton & Company, New York.

related to authentic mammalian

g

- than

g

S-crystallins

4. Chang, T., Jiang, Y.-J., Chiou, S.-H., and Chang, W.-C. (1988)

and may represent one intermediate crystallin form

Biochim. Biophys. Acta 951, 226 – 229.

from the divergent evolution of

g

-crystallin gene

fam-5. Pan, F.-M., Chang, W.-C., Chao, Y.-K., and Chiou, S.-H. (1994)

ily. On the other hand, shark

g

S-crystallins show only

_{Biochem. Biophys. Res. Commun. 202, 527 – 534.}

58-60% sequence identity to carp

g

S, underlying the

6. Pan, F.-M., Chang, W.-C., Lin, C.-H., Hsu, A.-L., and Chiou, S.-H. (1995) Biochem. Mol. Biol. International 35, 725 – 732.

distinct differences of

g

S-crystallins present in the

car-7. Quax-Jeuken, Y., Driessen, H. P. C., Leunissen, J., Quax, W., de

tilaginous and teleostean fishes.

Jong, W., and Bloemendal, H. (1985) EMBO J. 4, 2597 – 2602. 8. van Rens, G. L. M., Raats, J. M. H., Driessen, H. P. C.,

Old-enburg, M., Wijnen, J. T., Khan, P. M., de Jong, W. W., and

Construction of a Phylogenetic Tree Based on Protein

Bloemendal, H. (1989) Gene 78, 225 – 233.

Sequence Comparison

_{9. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) in Molecular}

Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold

We have previously constructed a phylogenetic tree

Spring Harbor, N.Y.

10. Chang, T., and Chang, W.-C. (1987) Biochim. Biophys. Acta 910,

of crystallins based on protein or DNA sequence

com-89 – 92.

parison with similar results (20 – 23). In Fig. 4 a

phylo-11. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl.

genetic tree is constructed based on multiple sequence

Acad. Sci. USA 74, 5463 – 5467.

alignment of eight protein sequences using a combina-

_{12. Hein, J. (1990) Methods Enzymol. 183, 626 – 645.}

tion of distance matrix and approximate parsimony

_{13. Siezen, R. J., Hom, C., Kaplan, E. D., Thomson, J. A., and}

methods (12). It has been shown to be useful to align

Benedek, G. B. (1988) Exp. Eye Res. 46, 81 – 93.

highly evolved gene families and their corresponding

14. Chiou, S.-H. (1989) FEBS Lett. 250, 25 – 29.

15. Chiou, S.-H., Chen, S.-W., Itoh, T., Kaji, H., and Samejima, T.

protein sequences that have clear evolutionary

rela-(1990) FEBS Lett. 275, 111 – 113.

tionships as in the cases of various crystallin family.

16. Smith, J. B., Yang, Z., Lin, P., Zaidi, Z., Abbasi, A., and Russell,

It is noteworthy that the phylogenetic tree based on

P. (1995) Biochem. J. 307, 407 – 410.

the sequence divergence among these protein se-

_{17. Blundell, T., Lindley, P., Miller, L., Moss, D., Slingsby, C., Tickle,}

quences indeed exemplifies the close relatedness be-

I., Turnell, B., and Wistow, G. (1981) Nature 289, 771 – 777.

tween shark

g

S-crystallins and

g

-crystallins from bo-

18. Wistow, G., Turnell, B., Summers, L., Slingsby, C., Moss, D.,

Miller, L., Lindley, P., and Blundell, T. (1983) J. Mol. Biol. 170,

vine and human lenses. On the other hand, carp

g

S-175 – 202.

crystallin was grouped with bovine

g

S-crystallin, in

19. Liaw, Y.-C., Chiou, S.-H., Lin, C.-W., Chang, T., and Chang,

agreement with the percent homology shown in Fig. 3.

_{W.-C. (1992) J. Biochem. 112, 341 – 345.}

Especially interesting is the observation that two

b

2-

_{20. Chiou, S.-H., Yu, C.-W., Lin, C.-W., Pan, F.-M., Lu, S.-F., Lee,}

crystallin sequences from bullfrog and bovine indeed

H.-J., and Chang, G.-G. (1995) Biochem. J. 309, 793 – 800.

21. Lu, S.-F., Pan, F.-M., and Chiou, S.-H. (1995) Biochem. Biophys.

locate at different branching points of the tree from

Res. Commun. 216, 881 – 891.

those of

g

- and

g

S-crystallins, indicative of two distinct

22. Pan, F.-M., Chang, W.-C., Lu, S.-F., Hsu, A.-L., and Chiou,

evolutionary pathways leading to

b

- and

g

/

g

S

crys-S.-H. (1995) Biochem. Biophys. Res. Commun. 217, 940 – 949.

tallins from the ancestral

b

/

g

protein.

_{23. Lu, S.-F., Pan, F.-M., and Chiou, S.-H. (1996) J. Protein Chem.}

15, 103 – 113.

24. Pan, F.-M., Chang, W.-C., Lu, S.-F., Hsu, A.-L., and Chiou, S.-H. (1995) Biochem. Biophys. Res. Commun. 217, 940 – 949.

ACKNOWLEDGMENTS

25. Hogg, D., Gorin, M. B., Heinzmann, C., Zollman, S., Mohandas, T. K., Klisak, I. J., Sparkes, R. S., Breitman, M. L., Tsui, L.-C., and Horwitz, J. (1987) Curr. Eye Res. 6, 1335 – 1341.

This work was supported by Academia Sinica and the National

26. Bhat, S. P., and Spector, A. (1984) DNA 3, 287 – 295. Science Council (NSC Grants 83-0203-B-001-086,

83-0418-B-001-020BA, 84-2311-B-001-050-BA & 86-2311-B-002-031-B15), Taipei, 27. Meakin, S. O., Du, R. P., Tsui, L.-C., and Breitman, M. L. (1987)

Mol. Cell. Biol. 7, 2671 – 2679.