Two Different Types of Interstitial Collagen in the Muscle Layer of the Marine Polychaete Hermodice sp.

Comp. Biochem. Physiol. Vol. 117B, No. 2, pp. 191–196, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0305-0491/97/$17.00 PII S0305-0491(96)00327-6

Two Different Types of Interstitial Collagen in the Muscle Layer of the Marine Polychaete Hermodice sp. Georgina Garza* and Alfonso Torre-Blanco Departamento de Biologıa´, Facultad de Ciencias, Universidad Nacional Auto´noma de Me´xico, Mexico City, Mexico

ABSTRACT. Two different interstitial collagens were isolated from the muscle layer of the marine polychaete worm Hermodice sp. (Annelida). Collagens were solubilized after limited pepsin digestion and fractionated by a differential salt precipitation procedure. Both types of interstitial collagen (Hermodice collagens A and B) appear to be homotrimers with subunit compositions (αA)3 and (αB)3 , respectively. Both types of subunits αA and αB migrate in SDS-PAGE between the α1(I) and α2(I) subunits of type I collagen with an apparent molecular weight of 100,000. SLS crystallites from Hermodice collagen A exhibited a band pattern similar to that of vertebrate type I collagen, whereas collagen B showed a very different band pattern, suggesting substantial sequence differences between the two types of collagen. The amino acid composition of Hermodice collagens A and B showed marked differences; notwithstanding, both collagens fit the pattern of interstitial collagens. The A :B ratio is 1.9, as determined from the amino acid analysis. comp biochem physiol 117B;2:191–196, 1997.  1997 Elsevier Science Inc. KEY WORDS. Annelid, collagen, evolution, invertebrate, collagen polymorphism, connective tissue

INTRODUCTION The presence of different types of collagen in animal tissues has been amply demonstrated in vertebrates. Indeed, during the last two decades, the collagen family of proteins has been growing steadily. At present, at least 19 different types of collagens have been described in vertebrate animals (17). Although much less comprehensively studied, invertebrates have also shown a great structural variability in their collagen molecules, some of them having no counterparts in vertebrates, like the cuticular collagens of annelids and nematodes (1,6). Even before the discovery of collagen polymorphism in vertebrates, it was known that annelids and nematodes possessed at least one additional collagen different from that present in the cuticle (3,4). Structural analysis of the collagen extracted from the intestines of the annelid Neanthes diversicolor showed it to be similar in size to vertebrate type I collagen (8). A type I-like interstitial collagen and a γ component probably derived from basement membrane collagen were isolated from octopus muscle (19). A more recent analysis of the collagens of the deep sea annelid Alvinella pompejana and the vestimentiferan tube worm Riftia pachyptila revealed, in addition to cuticle Address reprint requests to: A. Torre-Blanco, Depto. de Biologı´a, Facultad de Ciencias, UNAM, Me´ xico D.F. 04510, Mexico. Tel. (525) 622-4910; Fax (525) 622-4828. *Present address: Instituto de Fisiologı´a Celular, UNAM, Mexico City, Mexico. Received 31 May 1996; accepted 19 November 1996.

collagen, the presence of an interstitial collagen similar in some respects to fibril forming vertebrate collagens (5). Genetic analysis in the nematode Caenorhabditis elegans has revealed the presence of approximately 100 genes for cuticle collagen and two basement membrane (type IV) collagen genes (11), but no fibril forming collagen has been identified so far. Hermodice is a genus of marine polychaete worms closely related to the genus Nereis from which a cuticle collagen of the annelid type has been described (9,10,15). Here we describe the isolation of two different types of interstitial collagen from the muscle layer of the marine polychaetous annelid Hermodice sp. MATERIALS AND METHODS Specimens of Hermodice sp. were collected from the seashore of Isla de Sacrificios, near Veracruz, in the Gulf of Mexico. After collection, the specimens were maintained frozen at 218°C until use. Pepsin (twice crystallized, 2645 U/mg) and trypsin (type III, twice crystallized, 12,400 U/mg protein) were purchased from Sigma. Clostridium collagenase (349 U/mg) was obtained from Worthington, and electrophoresis reagents were from Bio-Rad. All other chemicals were of analytical grade. Acid-soluble guinea pig skin type I collagen was purified according to Torre-Blanco and Alvizouri (21), lyophilyzed and stored frozen at 218°C until use.

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Purification of Soluble Collagen From the Muscle Layer of Hermodice The worms were thawed and the muscle layer was dissected from the cuticle and the internal organs were put on an ice bath. The muscle layers from several worms were pooled and rinsed in cold distilled water. The insoluble material was collected by centrifugation at 34,000 g for 30 min, resuspended in 0.5 M acetic acid containing 0.025 M EDTA, 0.01 M N-ethylmaleimide and 0.001 M phenylmethanesulfonyl fluoride (PMSF) and stirred overnight at 4°C. The resulting suspension was centrifuged at 184,000 g for 30 min, the pellet was resuspended in 0.5 M acetic acid containing pepsin 0.2 mg/ml (final pepsin : tissue ratio was ,1 mg pepsin/g tissue), stirred overnight at 4°C and centrifuged at 184,000 g for 30 min. The supernatant was saved, and the pellet was further extracted overnight at 4°C in 0.5 M acetic acid containing 0.5 mg/ml pepsin. The suspension was centrifuged at 184,000 g for 30 min, the supernatants of both pepsin extractions were combined and dialyzed against several changes of 0.01 M Na2HPO4, pH 9.0; the precipitated collagen was collected by centrifugation at 1500 g for 90 min, redissolved in 0.5 M acetic acid and clarified at 184,000 g for 30 min. The resulting solution was precipitated by adding 4.0 M NaCl to give a final concentration of 1.0 M. The precipitate was collected at 1500 g for 30 min, redissolved in 0.5 M acetic acid and dialyzed against several changes of 0.5 M acetic acid; this unfractionated extract of purified Hermodice collagen was used for some experiments. The purified extract of soluble collagen was fractionated by a differential salt precipitation procedure in the following way. Enough volume of 4 M NaCl was added to the collagen solution, with continuous stirring, to take the salt concentration up to 0.6 M in a first step and up to 0.75 M in a second step. The precipitates were collected by centrifugation at 1500 g for 30 min and redissolved in 0.05 M acetic acid. The two collagen fractions obtained were further purified by ammonium sulfate precipitation; the fraction obtained at 0.6 M NaCl (Hermodice collagen A) precipitated at 15% saturation, whereas the fraction obtained at 0.75 M NaCl (Hermodice collagen B) precipitated at 20% saturation. Each fraction was dissolved in 0.5 M acetic acid and dialyzed against several changes of 0.5 M acetic acid. All steps of the purification procedure were carried out at 4°C or in an ice bath. Hydroxyproline Determination The concentration of collagen was estimated by measuring the hydroxyproline concentration. Samples were hydrolyzed in 6 N HCl, at 110°C, for 20 hr; HCl was eliminated by evaporation and the resulting amino acids were redissolved in water. The colorimetric procedure of Rojkind and Gonza´lez (18) was used for determining hydroxyproline in

tissues and crude extracts. The procedure described by Woessner (24) was used for determining hydroxyproline in purified collagen solutions.

Electrophoresis SDS-PAGE was performed according to Laemmli (12) in 7% polyacrylamide gels.

Digestion of Hermodice Soluble Collagen with Trypsin and Bacterial Collagenase Acid-soluble guinea pig type I collagen or Hermodice soluble collagen (unfractionated extract of purified Hermodice collagen) was dissolved in 0.005 M acetic acid to give a final concentration of 0.75 mg/ml. To neutralize each collagen solution, an equal volume of collagenase buffer (50 mM Tris–HCl, 0.15 M NaCl, 5 mM CaCl2, pH 7.6) was added. Trypsin or bacterial collagenase dissolved in collagenase buffer were added at a 20 : 1 substrate to enzyme ratio and the samples were incubated at 25°C; the reaction was stopped by addition of PMSF (1mM final concentration) to inhibit trypsin or EDTA (20mM final concentration) to inhibit collagenase. The extent of degradation was estimated after running the samples in SDS-PAGE.

Electron Microscopy Segment long spacing (SLS) crystallites from type I guinea pig skin collagen or Hermodice collagen was prepared by dialyzing collagen solutions in 0.1 M acetic acid against 0.4% ATP in 0.1 M acetic acid at 4°C overnight. After diluting the resulting suspension to a faint cloudiness, the crystallites were deposited on carbon-coated copper grids. Positive staining with phosphotungstic acid and uranyl acetate was performed according to Bruns and Gross (2). Negative staining with 2% sodium phosphotungstate was carried out as previously described (22). The grids were examined under a Zeiss EM 10 electron microscope and photographed at 50,0003; the exact magnification was determined with a cross-lined replica of a diffraction grating having 54,800 lines/inch (E. F. Fullam, Inc.).

Amino Acid Analysis The amino acid composition of Hermodice collagens A and B was kindly determined at the Protein Chemistry Facility, Washington University School of Medicine (St Louis, MO), by the method described by Moore and Stein (13). The analysis were carried out at two different sample concentrations to compensate for the high concentration of glycine. Methionine and cysteine were not determined. The ratios of polar, hydroxylic and hydrophobic amino acids

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were calculated using the amino acid analysis values expressed in residues/1000 according to Murray et al. (14). RESULTS Purification of Hermodice Soluble Collagen Pepsin limited digestion solubilized 22% of the hydroxyproline present in the muscle layer of Hermodice sp., whereas only traces were solubilized by 0.5 M acetic acid in the presence of proteinase inhibitors. The purification procedure described above rendered two fractions of soluble Hermodice collagen (called A and B) that were separated by their differential solubility. Denatured Hermodice collagens A and B had a similar electrophoretic pattern showing the presence of only one subunit migrating between the α1(I) and α2(I) subunits of type I collagen with an apparent molecular weight of 100,000 (Fig. 1). We refer to the subunits of Hermodice collagens A and B as αA and αB. The A : B ratio of the collagen fractions precipitated from the pepsinextracted collagen solution by the differential salt precipitation procedure was 1.9, as determined from the amino acid analysis data. Digestion by Trypsin and Bacterial Collagenase Trypsin digestion was done to ascertain the native state (i.e., the integrity of the triple helical structure) of the colla-

FIG. 2. (A) SDS-PAGE of Hermodice collagen incubated

with trypsin at different times. Lane 1, guinea pig type I collagen (shown for comparison); lane 2, 0 time; lane 3, 1 hr; lane 4, 2 hr; lane 5, 4 hr. (B) SDS-PAGE of Hermodice collagen digested by collagenase at different times. Lane 1, 0 time; lane 2, 1 hr; lane 3, 2 hr; lane 4, 4 hr; lane 5, 4 hr incubation with no enzyme added. The position of a subunits and b dimers has been indicated.

gen molecules. SDS-PAGE carried out after incubation in the presence of trypsin at 25°C showed that Hermodice collagen was not susceptible to proteolysis by this enzyme under the conditions assayed (Fig. 2A). On the other hand, Hermodice collagen was degraded by bacterial collagenase in a time dependent way (Fig. 2B); however, the rate of degradation of Hermodice collagen by collagenase was slower than that of guinea pig type I collagen (not shown). SLS Crystallites

FIG. 1. SDS-PAGE of Hermodice collagens A and B. (A) Left

lane, guinea pig type I collagen; right lane, Hermodice collagen A. (B) Left lane, Hermodice collagen B; right lane, guinea pig type I collagen. The position of a1(I) and a2(I) subunits has been indicated.

Positively stained SLS crystallites prepared from the unfractionated extract of purified Hermodice collagens showed to be a mixture of asymmetrical and centrosymmetrical crystallites with poorly defined band patterns, as well as headto-head or tail-to-tail dimeric crystallites and SLS fibers (results not shown). On the other hand, positively stained crystallites prepared from the fraction that precipitated at 0.6 M NaCl (Hermodice collagen A) showed a band pattern similar to that of type I collagen (Fig. 3, A and I). The SLS crystallites prepared from the fraction precipitated at 0.75M NaCl (Hermodice collagen B) (Fig. 3B) showed a band pattern clearly different to that of type I collagen or Hermodice collagen A. The average length of SLS crystallites from Hermodice collagen was 277 6 5 nm (mean 6 SD) as measured from negatively stained preparations (not shown).

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TABLE 2. Ratios of polar (R P), hydroxylic (R OH ) and hy-

drophobic (Rφ ) amino acids using amino acid analysis expressed as residues/1000

Hermodice A Hermodice B Invertebrate interstitial collagens†

Rφ

R OH

RP

185* 125*

375 352

439 523

140–226

296–403

418–521

*Met was not included. †Taken from Murray et al. (14). FIG. 3. Positively stained SLS crystallites of Hermodice col-

lagen A (A), guinea-pig type I collagen (I) and Hermodice collagen B (B).

Amino Acid Composition Table 1 shows the amino acid composition of Hermodice collagens A and B. The amino acid composition of Hermodice collagen A showed the typical characteristics of interstitial collagens as defined by the ratios of polar (Rp ), hydroxylic (ROH ) and hydrophobic (Rφ ) amino acids according to Murray et al. (14), whereas collagen B approached the limits established for interstitial collagens due to its high ratio of polar amino acids and its low ratio of hydrophobic amino acids (Table 2). Nevertheless, both Hermodice collagens showed an amino acid composition clearly different to basement membrane or annelid cuticular collagens.

TABLE 1. Amino acid composition of Hermodice collagens

A and B Amino acid Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine† Histidine Lysine Arginine

Collagen A*

Collagen B*

109.7 64.8 28.2 56.4 100.2 42.6 347.0 63.4 13.5 16.5 60.7 4.5 9.5 13.3 3.5 14.2 52.1

110.7 53.5 15.0 53.7 94.9 62.5 348.3 55.4 11.1 13.2 39.5 0 4.7 13.1 7.5 32.1 85.0

†The color factor for lysine was used. *Values are residues/1000 amino acid residues.

DISCUSSION The presence of interstitial collagen of the fibril forming type in the marine polychaete worm Hermodice sp. could be reasonably predicted. In fact, cross-striated collagen fibers typical of interstitial connective tissues have already been described in annelids (23), and an interstitial collagen of the fibril forming type has been isolated and thoroughly characterized from the annelid Alvinella pompejana (5). However, the finding of two different types of interstitial collagen in the muscle layer of Hermodice sp. was surprising. Although the presence of collagens of different types and functions (i.e., cuticular, basement membrane or interstitial) have been described in several invertebrates, there are no previous reports of interstitial collagen polymorphism such as it occurs in vertebrate tissues. The electrophoretic pattern of Hermodice interstitial collagens A and B showed only one α subunit migrating between the α1(I) and α2(I) subunits of type I collagen, with an apparent molecular weight of 100,000; this result indicates that both types of Hermodice collagens appear to be homotrimers with a subunit composition (αA)3 and (αB)3, respectively. The presence of either homo or heterotrimers of interstitial collagen have been described in different phyla of invertebrates (1,20), but there appear to be no rules that relate the subunit composition with established phylogenetic relationships in a simple way. Nevertheless, it is noteworthy that the interstitial collagen of the marine annelid A. pompejana showed an electrophoretic pattern similar to that of Hermodice interstitial collagens (5). The difficulty in solubilizing Hermodice collagens in the absence of pepsin suggests that they are extensively crosslinked, but future research is needed to determine the nature of these cross-links. Taking into account the fact that Hermodice collagen has been solubilized after pepsin limited digestion, we cannot rule out the possibility that putative globular extensions could have been destroyed by the enzymatic extraction procedure. On the other hand, this same fact indicates that the whole extension of the collagens isolated from Hermodice is of triple helical structure, as it was further confirmed by its resistance to trypsin digestion.

Annelid Interstitial Collagen Polymorphism

The band pattern of SLS crystallites reflects the distribution of clusters of charged and uncharged amino acids along the collagen molecule; accordingly, information on the primary structure can be obtained by comparing different patterns (2). The band patterns of SLS crystallites from Hermodice interstitial collagens A and B were clearly different, indicating substantial sequence differences between them. On the other hand, Hermodice collagen A exhibited a band pattern remarkably similar to that of guinea pig type I collagen, whereas the band pattern of Hermodice collagen B appeared to be unique. SLS crystallites with a unique band pattern, different from that of type I collagen, have been described in platyhelminths, annelides and vestimentiferans (5,22,23), whereas segments with a band pattern similar to that of vertebrate type I collagen have also been described in platyhelminths as well as in coelenterates (7,16). The conservation of the distribution of charged amino acid clusters during the evolution of such different phyla is a remarkable feature of these interstitial collagens, suggesting a crucial role in fibril formation, but still many differences in amino acid sequence may be present in collagens exhibiting the same band pattern. The amino acid composition of both interstitial collagens from Hermodice sp. fits the pattern of interstitial collagens as defined by the ratios of polar, hydoxylic and hydrophobic amino acids (14), although the composition of collagen B approaches the limits established for interstitial collagens. Nevertheless, the differences in amino acid composition clearly differentiate collagen A from B; most notably, the content of basic amino acids in Hermodice collagen A is much lower (about 60%), whereas its content of hydrophobic amino acids is higher than that of collagen B (about 153%). In this respect, Hermodice collagen B resembles the interstitial collagen of the marine annelid A. pompejana more than Hermodice collagen A, but still there are important differences between the amino acid composition of these collagens. Despite the similarity in SLS band patterns between Hermodice collagen A and vertebrate type I collagen, their amino acid compositions do not appear to be closely related. From the amino acid analysis data, we could also estimate the A: B ratio of the collagen fractions; this result indicated that the amount of collagen A is nearly twice relative to collagen B. Hermodice collagen was resistant to proteolysis by trypsin and was degraded by bacterial collagenase, as expected from collagen molecules without interruptions in their triple helical structure. However, its rate of hydrolysis by collagenase was slower than for type I collagen. At present we do not have an explanation for this phenomenon. The presence of interstitial collagen polymorphism in the muscular tissue of the marine annelid Hermodice sp. suggests that the extracellular matrix of these animals is probably more complex than previously thought and that their collagen fibrils could be formed by the aggregation of more than

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one collagen type, as has been shown to occur in the tissues of vertebrates. We gratefully acknowledge Dr. Annie Pardo and Dr. Eugene A. Bauer for the amino acid analysis.

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