Annelid skin collagen: Occurrence of collagen with structure of (α1)2α2 in Urechis unicinctus

Comp. Biochem. Physiol. Vol. 75B, No. 4, pp. 681-684, 1983 Printed in Great Britain

0305-0491/83 $3.00+0.00 © 1983 Pergamon Press Ltd

ANNELID SKIN COLLAGEN: OCCURRENCE OF COLLAGEN WITH STRUCTURE OF (0~1)2~2 IN URECHIS UNICINCTUS SHIGERU KIMURA,* HIROYUKI TANAKA* and YEUNG-Ho PARKt *Laboratory of Biochemistry, Tokyo University of Fisheries, Minato-ku, Tokyo 108, Japan and tFood Science and Technology, National Fisheries University of Busan, Nam-gu, Busan, Korea (Received 13 January 1983) Abstract--1. The skin collagen of an annelid, Urechis unicinctus, was found to have a chain composition of (2 1)2~2. 2. This Urechis skin collagen, as well as octopus skin collagen reported previously, was assumed to be homologous to vertebrate skin Type I collagen. 3. Thus, we propose the use of the term Type I collagen to describe the homologous protein derived from protostomian and deuterostomian animals.

INTRODUCTION Invertebrate collagens may be subdivided into three groups on the basis of molecular weight and subunit composition; (1) interstitial collagens comprising chain-sized components, (2) basement membrane collagens (Hung et al., 1980, 1981; Takema and Kimura, 1982), and (3) cuticle collagens (Josse and Harrington, 1964; Kimura and Tanzer, 1977; Ouazana and Herbage, 1981). Some interstitial collagens are characterized by having three identical ~ chains (Ashhurst and Bailey, 1980; Francois et al., 1980; Katzman and Kang, 1972; Kimura and Tanaka, 1983). On the other hand, our previous studies have clearly shown the occurrence in octopus collagen of two distinct ~ chains, ~ 1 and ~ 2, which are homologous to those in Type I collagen of higher vertebrates (Kimura and Matsuura, 1974; Kimura et al., 1981b; Takema and Kimura, 1982). Thus, we have proposed that Type I or Type I-like collagen evolved along independent phylogenetic lines of Protostomia and Deuterostomia (Kimura et al., 1981b). Although the possible presence of collagen with an (~ 1)2~2 composition in Acanthocephala (Cain, 1970), Echinodermata (Pucci-Minafra et al., 1978), and Platyhelminthes (Torre-Blanco and Toledo, 1981) has been suggested by SDS-polyacrylamide gel electrophoresis, little definitive information is available concerning the distribution of the homologous collagen in the protostome line. In the present study, the skin tissue of an annelid, Urechis unicinctus, was shown to contain a collagen homologous to Type I collagen. MATERIALS AND METHODS Preparation of skin collagen Large UrechLv unicinctus were collected in Korea and transported to the laboratory in Tokyo. Urechis skin collagen was isolated essentially as described previously (Kimura et al., 1981b). All operations were performed in a Abbreviation: SDS, sodium dodecyl sulfate. cap

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cold room at 3-5°C. The skin tissue was cleaned off soft tissues, cut into small pieces, and extracted with successive changes of 0.5 M sodium acetate and 0.5 M acetic acid for several days. Only a negligible amount of the soluble collagen was obtained by these extractions. Therefore, the residual tissue (20 g wet wt) was solubilized by limited proteolysis with pepsin (Sigma, 2 × ; 100 mg) in 0.5 M acetic acid for 24 hr at 4°C. Approximately 80~ of the tissue collagen was rendered soluble and the digestion mixture was clarified by centrifugation at 10,000g for 1 hr. The collagen present in the supernatant was precipitated by addition with stirring of 4.0 M NaC1 to 0.40 M. The precipitate harvested by centrifugation was washed with 0.02 M Na2HPO 4 for 24 hr in order to inactivate the pepsin and then dissolved in 0.05 M Tris-HCl, pH 7.5, containing 1 M NaC1. To the collagen solution was added crystalline NaC1 to a final concentration of 3.2 M. The resulting precipitate was redissolved in 0.5 M acetic acid, dialyzed against 0.1 M acetic acid, and lyophilized. The yield of collagen was about 200 mg. Analytical methods SDS-polyacrylamide gel electrophoresis, CM-cellulose chromatography, Sepharose CL-4B molecular sieve chromatography, CNBr-peptide mapping, formaldehyde treatment and amino acid analysis of the isolated skin collagen and/or its ~ chains were performed essentially as described (Kimura et al., 1981b). RESULTS Chain composition o f skin collagen The collagen fibers of Urechis skin were susceptible to limited proteolysis with pepsin and easily solubilized into solution in the form of native molecules. Electrophoresis in SDS of the solubilized collagen (Fig. 1, 0) revealed the presence of two major components in the region of ~ chains; the upper band was designated c~1 and the lower one ~2. In addition, there were minor bands corresponding to crosslinked components, fl and y chains, and two additional components migrated further than ~2. The latter two components may be derived from degradation products of the c~ chains, because they could not be re-

682

SHIGERU KIMURA et al. Table 1. Amino acid composition of Urechis skin collagen and its subunits (residues/1000) 3-Hyp 4-Hyp Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe Hyl Lys His Arg Glc-Gal-Hylt

0

1

2

3

Fig. 1. SDS-polyacrylamide gel electrophoresis of pepsinsolubilized collagen and its chromatographic fractions from Urechis skin. The original collagen and its fractions indicated by numbers in the CM-cellutose chromatogram (Fig. 2) were resolved on 3.5% polyacrylamide gels containing 0.1~o SDS, 3.5 M urea and 0.1 M sodium phosphate pH 7.2. 0: Original, 1: Fraction 1, 2: Fraction 2, 3: Fraction 3.

m o v e d by differential salt fractionation o f the native collagen u n d e r various conditions. F u r t h e r m o r e , reduction of the collagen with dithiothreitol did not affect the electrophoretic patterns, indicating the absence of disulfide bonds. The constituent ~ chains were isolated by a combin a t i o n o f CM-cellulose c h r o m a t o g r a p h y a n d molecular sieve c h r o m a t o g r a p h y on Sepharose CL-4B. T h e d e n a t u r e d skin collagen was initially fractionated on CM-cellulose as s h o w n in Fig. 2; it was separated into

I

0.8

Collagen

~1

~2

7H2'

5 53 76 29 44 93 93 345 96 15 16 10 17 4 6 11 12 9 66 5

5 60 75 27 40 94 97 347 95 14 18 7 18 3 6 9 12 7 66 3

5 41 76 31 49 94 89 354 99 16 12 14 13 4 6 10 10 10 67 5

5 55 77 29 43 94 94 345 96 15 16 10 16 3 6 10 11 9 66 4

*Obtained by CM-cellulose chromatography of formaldehyde-treated collagen. tGlucosylgalactosylhydroxylysine. two fractions each of which c o n t a i n e d an ~ chain as a m a j o r c o m p o n e n t . W h e n e x a m i n e d by SDS-gel electrophoresis (Fig. 1, 1-3), the first peak corres p o n d i n g to the :t 1 a n d the second one the ~2. It is

0~I

62

o~I

o~2

I

I

I

C/d

i 0.4 I

I

CALF o~

I

I

150 300 ELUTION VOLUME (ml)

Fig. 2. CM-cellulose chromatography of Urechis skin collagen. The column, 1.5 x 10cm, was equilibrated with 0.01 M sodium acetate, pH 4.8, containing 4 M urea and maintained at 30°C. Samples were eluted with a linear gradient of 0-0.08 M NaCI over a total volume of 800 ml at a flow rate of 120 ml/hr.

URECHIS

Fig. 3. SD~gel etectrophoresis of CNBr-peptides derived from ~1 and ~2 chains of Urechis skin collagen. The CNBr-peptides were resolved on 10~o polyacrylamide gels containing 0.1~o SDS and 0.1 M sodium phosphate, pH 7.2. The ~ 1 and ~2 chains of calf skin Type I collagen were digested with CNBr and examined in parallel with the Urechis • chains. The major peptides derived from calf skin chains are identified; the numbers 8 and 4 mean ~ 1(1)CB-8 and ~2(I)CB-4, respectively.

Annelid skin collagen noteworthy that small but significant amounts of two different fl chains, presumably flll and fl12, were found to be present in the ~1 peak and the shoulder preceding the ~2, respectively. This chromatographic pattern appears to be characteristic of that for Type I or Type I-like collagen (Kimura and Matsuura, 1974; Kimura et al., 1981a, b; Piez et al., 1963). Furthermore, the proportion of ~ 1 and ~2 chains was calculated to be approximately 2:1, suggesting that the possible chain composition of Urechis skin collagen may be designated (ct 1)2~2. In order to isolate the ~ chains, each protein recovered from the respective peaks was then chromatographed on Sepharose CL-4B (data not shown). The major fraction corresponding to an ~ component was pooled, desalted and lyophilized. The apparent molecular weights of ~ 1 and ~2 chains were estimated to be approximately 99,000 and 88,000, respectively, on the basis of their elution positions. The results of amino acid analyses are presented in Table 1. The overall amino acid compositions of ~ 1 and ~t2 were similar to those from octopus skin collagen (Kimura et al., 1981b) homologous to vertebrate Type I collagen. The ctl and ~2 chains resembled each other I

I

683

with the exception of elevated levels of 4-hydroxyproline, methionine and leucine and a reduced level of isoleucine in the ~1. Furthermore, the original collagen was found to be consistent with a 2:1 mixture of 1 and ~2 chains; this may support the chain composition of (~tl)2~2 for Urechis skin collagen. Incidentally, the ~t2 was unique in a higher content of isoleucine than leucine; this feature has been reported only in a collagen from elastoidin of shark fins (Kimura and Kubota, 1966). Both ~t chains were further examined by CNBr digestion followed by SDS-gel electrophoresis. Figure 3 clearly indicates the non-identity of ctl and ~2 chains. It also shows that the CNBr peptides of the subunits of Urechis skin collagen differ from those of well-characterized calf skin Type I collagen. Characterization o f Jbrmaldehyde-treated collagen

The proposed chain composition of (~1)2ct2 for Urechis skin collagen was confirmed by introducing intramolecular crosslinks into native molecules, according to procedures described previously (Kimura et al., 1981b). The formaldehyde-treated collagen, after denaturation, was chromatographed on CMI

I

0,9 A

B

E

c-

o

oil

0,6

v

i,i z Q:: C)

0,3

0

B

I

I

5O

i00

I

150

I

200

ELUTION VOLUME (ml) Fig. 4. CM-cellulose chromatography of formaldehyde-treated Urechis skin collagen. A 0.02~o solution of Urechis skin collagen in 0.1 M acetic acid was treated with 3.4% formaldehyde at 4°C for 3 days. The resulting crosslinked collagen was resolved on a column, 0.9 x 7 cm, equilibrated with 0.01 M sodium acetate, pH 4.8, containing 4 M urea at 30°C. Samples were eluted with a linear gradient of 0~.08 M NaC1 over a total volume of 400ml. The arrows indicate the elution positions of Urechis ~ 1 and ~2 chains. Insets show SDS-gel electrophoresis of Urechis skin collagen before and after formaldehyde-treatment. A: Original collagen; B: Formaldehyde-treated collagen.

684

SH1GERU KIMURAet al.

cellulose as a single peak at a position between • 1 and • 2 peaks (Fig. 4). The protein recovered from the peak was pooled and examined by SDS-gel electrophoresis; it comprised largely a ~ chain (Fig. 4). Furthermore, the protein exhibited an amino acid composition almost identical to a mixture of two ~ 1 chains and one ~2 chain as shown in Table 1. Thus, the 7 chain was reasonably identified as 7,~2 with structure of(c~ 1)2 c~2 on the basis of its elution position and amino acid composition, as was the case with octopus skin collagen (Kimura et al., 1981b).

Morphological and biochemical characterization. Eur. J. Biochem. 103, 75-83.

Bornstein P. and Sage H. (1980) Structurally distinct collagen types. A. Rev. Bioehem. 49, 957 1003. Cain G. (1970) Collagen from the giant acanthocephalan Macracanthorhynchus hirudinaceus. Archs biochem. Biophys. 141, 26zb270.

Francois J., Herbage D. and Junqua S. (1980) Cockroach collagen: Isolation, biochemical and biophysical characterization. Eur. J. Biochem. 112, 389-396. Hung C.-H., Butkowski R. J. and Hudson B. G. (1980) Intestinal basement membrane of Ascaris suum: Properties of the collagenous domain. J. biol. Chem. 255, 4964-4971. DISCUSSION Hung C.-H., Noelken M. E. and Hudson B. G. (1981) Intestinal basement membrane of Ascaris suum: Physical An annelid skin collagen has been isolated from properties of the collagenous domain. J. biol. Chem. 256, Urechis of the class Echiuroidea and was found to 3822-3826. markedly contrast in molecular properties with well- Josse J. and Harrington W. F. (1964) Role of pyrrolidine established cuticle collagens from Lumbricus and N e residues in the structure and stabilization of collagen. J. reis of the classes Oligochaeta and Polychaeta, remolec. Biol. 9, 269-287. spectively, of the same phylum (Josse and Harrington, Katzman R. L. and Kang A. H. (1972) The presence of fucose, mannose and glucosamine-containing hetero1964; Kimura and Tanzer, 1977). Characterization of polysaccharide in collagen from the sea anemone Metthe solubilized collagen revealed that it had the same ridium dianthus. J. biol. Chem. 247, 5486-5489. chain composition of (~ 1)2~2 as calf skin Type I collagen, while CNBr-peptide maps of both collagen ~ 1 Kimura S. (1983) Vertebrate skin Type I collagen: Comparison of bony fishes with lamprey and calf. Comp. or c~2 chains were different. Moreover, the Urechis Biochem. Physiol. 74B, 525-528. skin ~ chains were similar in their amino acid com- Kimura S. and Kubota M. (1966) Studies of elastoidin--l. position to ~ chains of Type I collagen rather than Some chemical and physical properties of elastoidin and those of Types I I I - V collagens. In addition to these its components. J. Biochem., Tokyo 60, 615-621. results, the fact that the major collagenous protein of Kimura S. and Matsuura F. (1974) The chain composition of several invertebrate collagens. J. Bioehem., Tokyo 75, vertebrate skin tissues is classified as Type I collagen 1231-1240. (Kimura, 1983) strongly suggests that the Urechis skin collagen is homologous to Type I collagen, because a Kimura S. and Tanaka H. (1983) Characterization of top shell muscle collagen comprising three identical c~1 chains. characteristic anatomical distribution is considered to Bull. Jap. Soc. scient. Fish. 49, 229-232. be an important factor in the type classification of Kimura S. and Tanzer M. L. (1977) Nereis cuticle collagen: collagen. Thus, it is very probable that a protein hoIsolation and characterization of two distinct subunits. mologous to Type I collagen is distributed in a wider Biochemistry, N.Y. 16, 2554~2560. range of protostomian animals; at present the occur- Kimura S., Kamimura T., Takema Y. and Kubota M. rence of the homologous collagen has been confirmed (1981a) Lower vertebrate collagen: Evidence for Type I-like collagen in the skin of lamprey and shark. Biochim. in the two different phyla, Annelida and Mollusca. biophys. Acta 669, 251 257. This finding is compatible with our view that Type I or Type I-like collagen evolved along independent phy- Kimura S., Takema Y. and Kubota M. (1981b) Octopus skin collagen: Isolation and characterization of collagen logenetic lines of Protostomia and Deuterostomia comprising two distinct :~ chains. J. biol. Chem. 256, (Kimura et al., 1981b). 13230-13234. On the basis of these composite results, we propose, Ouazana R. and Herbage D. (1981) Biochemical characterby definition, the use of the term Type I collagen to ization of the cuticle collagen of the nematode Caedescribe the homologous protein derived from protonorhabditis elegans. Bioehim. biophys. Acta 669, 236-243. stomian and deuterostomian animals, since we refer to Piez K. A., Eigner E. A. and Lewis M. S. (1963) The chromatographic separation and amino acid composition homologous proteins within the collagen family as of the subunits of several collagens. Biochemistry, N. Y. 2, Types (Bornstein and Sage, 1980). 58 66. Pucci-Minafra I., Galante R. and Minafra S. (1978) Acknowledgements This work was supported by a grant Identification of collagen in the Aristotle's lanternae of from the Ministry of Education, Science and Culture of Paracentrotus lividus. J. submicr. Cytol. 10, 53-63. Japan (1982). Takema Y. and Kimura S. (1982) Two genetically distinct molecular species of octopus muscle collagen. Biochim. biophys. Aeta 706, 123 128. Torre-Blanco A. and Toledo I. (1981) The isolation, REFERENCES purification and characterization of the collagen of Cysticercus cellulosae. J. biol. Chem. 256, 5926-5930. Ashhurst D. E. and Bailey A. J. (1980) Locust collagen: