Studies on the sulfhydryl groups in type III collagen

Bioehimica et Biophysica Acta, 446 (1976) 240-244

© Elsevier/North-Holland Biomedical Press BBA 37441 STUDIES ON T H E S U L F H Y D R Y L G R O U P S IN TYPE III C O L L A G E N

MICHAEL SCHNEIR" and EDWARD J. MILLER Department of Biochemistry and lnstitute of Dental Research, University of Alabama Medical Center, Birmingham~ Ala. 35294 (U.S.A.)

(Received March 23rd, 1976)

SUMMARY The Type lII collagen molecule, [al(III)]3, is comprised of three al(III) chains each of which contains two cysteinyl residues. Free sulfhydryl groups, however, could not be detected in the denatured, trimeric ),-component of Type Ili collagen as judged by the failure to form derivatives with the alkylating reagents iodo[14C]acetic acid and 4-vinylpyridine. This absence of sulfhydryl group reactivity did not appear to result from disulfide-binding between Type III collagen and noncollagenous peptides. Collectively, the results indicate that all of the six sulfhydryl groups of the Type III collagen molecule participate in interchain disulfide-bonding.

INTRODUCTION After denaturation, the majority of the protein in Type III collagen preparations from dermis is recovered as F-components with a molecular weight of 300 000 [1-6]. The ),-components are formed as the result of interchain disulfide-binding, since F-components are converted to al(III) chains (100 000 daltons) by reduction with 2-mercaptoethanol or dithiothreitol. The al(III) chain contains two cysteinyl residues and at least four of the available six sulthydryl groups would be involved in interchain disulfide-bonding leading to the formation of the trimeric ),-component. The disposition of the remaining two sulfhydryl groups is currently unknown and was the subject of the studies reported here. MATERIALS AND METHODS Preparation of collagen

Type III collagen was obtained by selective salt-precipitations of pepsinsolubilized infant dermal collagen as previously described [1]. After denaturation, disulfide-bonded ),-components were obtained from this collagen preparation by molecular sieve chromatography [3] of 20-50 mg quantities on a 1.5 x 155 cm column of Studies were performed while on sabbatical leave from the Department of Biochemistry, University of Southern California School of Dentistry, Los Angeles, California 90007. Send correspondence to this address.

241 Sepharose 4B (Pharmacia). al(III) chains were obtained by reduction of the ?-components with 2-mercaptoethanol and rechromatography on Sepharose 4B [3]. The cysteine-containing cyanogen bromide-peptide of al(III), al(III)-CB9, was isolated in trimer form by carboxymethyl cellulose chromatography as previously described [3].

Alkylation with Iodoacetic acid Samples of 7-components were reduced with 2-mercaptoethanol and alkylated with iodo[14C]acetic acid essentially as previously described [1]. For alkylation in the absence of reducing agent, the protein samples were dissolved at a concentration of 7-10 mg/ml in 5.0 M deionized urea (pH 8.0) containing 0.36 M iodoacetic acid (sodium salt) and sufficient iodo[14C]acetic acid (250#Ci) (New England Nuclear) to attain a specific activity of 0.6 Ci/mol of iodoacetate. The alkylation reaction was allowed to proceed for one hour in the dark at room temperature. After reduction and alkylation or alkylation alone, the reaction mixtures (2 ml) were applied directly to Sepharose 4B for chromatography and recovery of the ),-components and al(III) chains. Appropriate fractions of the column effluent were desalted on a column of Bio-Gel P-2 (equilibrated with 0. l M acetic acid) and lyophilized.

Alkylation with 4-vinylpyridine The 7-components were reduced with 2-mercaptoethanol and alkylated with 4-vinylpyridine using previously described procedures [7] with appropriate modifications. These included reduction of the quantity of sample to 10 mg and a 100-fold reduction in the volume of all reagents. For alkylation in the absence of reducing agent, samples were dissolved at a concentration of 10 mg/ml in the alkylation solution and the reaction was allowed to proceed for 2 h. Following reduction and aikylation or alkylation alone, the reaction mixtures (I-2 ml) were applied directly to Sepharose 4B for chromatography and recovery of the 7-components and al(III) chains.

Analysis for bound cysteine-containing peptides After reduction. Samples of ?-components (30-50 rag) were reduced with 2mercaptoethanol [1] and the reaction mixtures applied to a 2.5 x 40 cm column of Bio-Gel P-2 (Bio-Rad Laboral~ories) equilibrated with 0.1 M acetic acid. The fractions of the Bio-Gel P-2 column effluent eluted after the void-volume of the column were collected, adjusted to pH 1.5 with HC1, and applied to a 3 × 15 cm column of Dowex 50 (AG 50W-X2, 100-200 mesh, Bio-Rad Laboratories) in the ammonium form. The column was then washed with five volumes of distilled water and eluted with 2.0 M ammonium acetate (pH 7.0). The ammonium acetate eluate was lyophilized, hydrolyzed, and approximately two-thirds of the hydrolysate applied to the analyzer column. Similar experiments were performed with trimeric al(III)-CB9 (45 rag) with the exception that the reduced protein was applied to the 1.5 x 155 cm Sepharose 4B column. Fractions corresponding to the total fluid volume of the column were lyophilized and applied to a Bio-Gel P-2 column. Fractions corresponding to the voidvolume and the remaining column effluent from the latter column were collected, lyophilized, and hydrolyzed for amino acid analyses.

242

After performic acid oxidation. Samples of ),-components were oxidized by performic acid as described previously [3]. After lyophilization, the samples were chromatographed on Bio-Gel P-2 and appropriate fractions (as discussed above) were lyophilized and subsequently hydrolyzed for amino acid analyses. Amino acid analyses Protein samples were hydrolyzed in triply-distilled HC1 at 104 °C for 24 h. Amino acid analyses were performed on an automatic amino acid analyzer as previously described [11 ]. Under the conditions employed, S-(4-pyridylethyl) cysteine eluted between ammonia and arginine at approximately 285 min. The resolution of Scarboxymethyl cysteine, which eluted between hydroxyproline and aspartic acid, was enhanced by decreasing the pH of the initial buffer to 2.8. In certain experiments, the analyzer column effluent was collected in 1.5 ml fractions and 0.5 ml aliquots were counted in a liquid scintillation counter (Beckman, Model LS-233) after addition of 4 ml of Aquasol (New England Nuclear). RESULTS Amino acid analyses of Type III collagen y-components after reaction with iodoacetic acid or 4-vinylpyridine in the absence of reducing agent revealed that the respective derivatives, S-carboxymethyl cysteine and S-(4-pyridylethyl) cysteine, were not present in the hydrolysates. Indeed, neither of these derivatives were detectable when as much as 5.0 mg of protein (0.017 #tool) was hydrolyzed and applied to the analyzer column. Assuming that two sulfhydryl groups per y-component would theoretically be available for reactivity with the alkylating reagents, samples of this size would certainly have been sufficient to detect the derivatives by the method employed. Nevertheless, an even more sensitive assay utilizing iodo[14C]acetic acid was employed. As indicated in Fig. 1, the portion of the analyzer effluent corresponding to the elution position of S-carboxymethyl cysteine was devoid of radioactivity when alkylation was attempted in the absence of reducing agent. In contrast to the above results, alkylation of sulfhydryl groups occurred readily in the presence of reducing agent. Amino acid analysis of the isolated al(III) chains following reduction and alkylation showed the presence of approximately 1.7 and 1.2 residues per 1000 amino acid residues of S-carboxymethyl cysteine and S-(4-pyridylethyl) cysteine, respectively. As expected, under these conditions, radioactivity was readily detectable in the S-carboxymethyl cysteine peak eluted from the analyzer column (Fig. 1). The lack of sulfhydryl group reactivity for Type III collagen y-components could not be ascribed to disulfide-bonding of the y-components to smaller cysteinecontaining peptides. Small amounts of low molecular weight peptides could be recovered after reduction of y-components and trimeric al(III)-CB9 as well as after performic acid oxidation of y-components. However, in each experiment amino acid analysis of these peptides showed the presence of far less cysteine or cysteic acid (3-5 ~) than would be required for disulfide-bonding to two cysteinyl residues of the y-components or trimeric al(III)-CB9.

243 ¢J

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Fig. 1. Radioactivity pattern in the amino acid analyzer column effluent: ( 0 - - 0 ) hydrolysate of 0.5 mg of Type III collagen V-componentsexposed to iodo[14C]aceticacid in the absence of reducing agent; (O--41) hydrolysate of 0.5 mg of al(III) chains following exposure of Type III collagen ycomponents to iodo [J4C]aceticacid in the presence of reducing agent. Arrows indicate the elution positions of hydroxyproline Pro(OHm,S-carboxymethyl cysteine (CMC), and aspartic acid (Asp). DISCUSSION The potentially free sulfhydryl groups in the ),-component of Type III collagen were found to be unreactive to two alkylating reagents, i.e., iodoacetic acid and 4vinylpyridine. In data not presented, we also found that these groups were unreactive to 5,5-dithiobis (2-nitrobenzoic acid) [9] and 2,2'-dithiodipyridine [10]. Furthermore, the },-components could not be adsorbed to thio-activated SH-Sepharose 4B (Pharmacia) when adsorption was performed by the method recommended for mercaptalbumin Ill]. Perhaps the most convincing evidence that the },-component is devoid of free sulfhydryl groups is the failure to form a detectable derivative during reaction with iodo[l¢C]acetic acid in the absence of reducing agent. All attempts to isolate smaller cysteine-containing peptides after reduction or performic acid oxidation of },-components or trimeric a l (III)-CB9 were unsuccessful. These results, then, strongly suggest that all cysteine residues of the Type III collagen },-component participate in interchain disulfide-bonding. Further, it would appear that solubilization of Type III collagen during limited pepsin-digestion does not occur as the result of cleavage of noncollagenous proteins which are disulfide-bonded to Type III collagen molecules. It is, perhaps, understandable that all of the sulfhydryl groups in the },-components and presumably the native Type III collagen molecule would be disulfidebonded. The presence of both free sulfhydryl groups and disulfide-bonds in the same protein is relatively rare [12]. Mercaptalbumin, however, does contain a potentially free sulfhydryl group, but it is for the most part utilized in disulfide-bonding to additional small peptides [13]. In addition, it would seem undesirable for an extraceUular protein to contain reactive free sulfhydryl groups. In this regard, the cysteine-containing collagenous components isolated from glomerular basement membrane [14], Ascaris cuticle [15], and Ascaris muscle [16] are reported to be devoid of free sulfhydryl groups. Preparations of Type III collagen often contain some aggregates of al(III) chains with molecular weights greater than that of the 7-component [3]. This obser-

244 vation along with the present results suggest that in situ some disulfide-interchange m a y occur leading to the f o r m a t i o n o f i n t e r m o l e c u l a r disulfide cross-links. It is also p r o b a b l e , however, that the higher m o l e c u l a r weight c o m p o n e n t s are f o r m e d t h r o u g h disulfide-interchange during isolation a n d purification o f the T y p e I I I collagen. Similarly, the existence o f interchain disulfide-bonding in isolated [al(III)]3 c o u l d arise either in situ or during the course o f the isolation procedure. F u r t h e r w o r k will be required to establish the significance o f b o t h interchain a n d i n t e r m o l e c u l a r disulfideb o n d i n g in stabilizing the molecules o f T y p e I I I collagen within fibers. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by G r a n t s DE-02670 a n d DE-03318 from the N a t i o n a l Institute o f D e n t a l Research, U . S . P . H . S . W e t h a n k Dr. K e n t R h o d e s , Virginia Wright, a n d Elizabeth Keele for p e r f o r m i n g a m i n o acid analyses. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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