Collagen Rel. Res. Vol. 1/1981, p. 543-548

Peptide Mapping of Collagen Chains U sing CNBr Cleavage of Proteins Within Poly acryl amide Gels

GREGORY s. BARSH, KAREN E. PETERSON and PETER H. BYERS

Departments of Pathology and Medicine, University of Washington, Seattle, WA 98195, USA.

Abstract

A peptide mapping technique is described which uses a two dimensional format to display CNBr peptides of collagen chains. Biosynthetically-labeled products from 250,000 cells are analyzed in each map by a rapid procedure which does not require preliminary purification steps. Pro teins trapped within polyacrylamide gels are digested with CNBr under conditions where diffusion of radiolabeled peptides from gels is negligible, and the reaction products are recovered quanti­tatively by electroelution.

Peptide maps of pro (J. chains, a chains, and TCA chains cleaved with mam­malian collagenase are presented with the identification of specific fragments. This method is useful for the analysis of structural abnormalities in collagen pro teins from patients with certain genetic dis orders, examination of collagenous proteins produced by primary cultures which exhibit phenotypic switching, and identification of new collagen types.

Key words: Peptide mapping, polyacrylamide gels, collagen

Introduction

During the last ten years, it has been recognized that the collagen gene family includes at least six related oligomeric pro teins coded for by at least nine separate genes (Bornstein and Sage, 1980). Although many of these collagens were identified by biochemical studies of highly processed and crosslinked polymers in tissues, structural characterization of new collagen types and of genetic variants present in heritable disorders has been greatlyaided by the analysis of pro collagens produced by cells in culture (Crouch et al., 1980; Bornstein and Byers, 1980; Tryggvason et al., 1980). Cleavage with CNBr or mast protease has been used to analyze structurally distinct collagen chains, but these techniques generally require several preliminary steps of purifcation and, consequently, relatively large amounts of starting material (Rhodes and Miller, 1979; Sage et al., 1979).

We have developed a rapid, sensitive, and inexpensive peptide mapping tech­nique using CNBr cleavage oi proteins within polyacrylamide gels. This technique has been used to identify a structural variant of pro a 1(I) chains! produced by cells from a patient with lethai perinatal osteogenesis imperfecta (Barsh and Byers,

544 G. S. Barsh, K. E. Peterson and P. H. Byers

1981), and to localize an abnormality in an cx 2(1) chain produced by a patient with a form of the Marfan syndrome (Byers et al., in press). The procedure and its applications are detailed in this communication.

Materials and Methods

Cell Culture, Radioisotope Labeling, and Enzyme Digestions

Procedures used for cell culture were described elsewhere (Barsh and Byers, 1981).

Briefly, human dermal fibroblasts were plated at a density of 250,000 cellsl 35 mm culture dish and allowed to attach and spread for 24 h. Each culture was then incubated with 0.5 ml of Dulbecco-Vogt-modified Eagle's medium which contained 50 ,ug/ml of ascorbic acid and 200 ,uCi/ml of L-(2,3-3H) proline (25 Cil mmole, New England Nuclear). After a 16 h incubation, medium proteins were harvested in the presence of protease inhibitors (Barsh and Byers, 1981) and then dialyzed against 0.5 N acetic acid (for digestion with pepsin), or prepared for electrophoresis. Cell layer proteins can be prepared in a similar manner for analysis of intracellular procollagens (Barsh and Byers, 1981).

Pepsin digestions were performed overnight at 4 °C with 50 ,ug/ml of pepsin (Worthington) an 24 ,ug/ml carrier human type I collagen (a gift from Dr. Gary Striker). Reactions were terminated by addition of pepstatin A to a final con­centration of 5 ,ug/ml, and the mixt ure was then dialyzed against 10 mM calcium chloride and 50 mM Tris, pH 7.5 (for digestion with with mammalian colla­genase), or prepared for electrophoresis.

Digestion with mammalian collagenase (generously provided by Dr. Eugene Bauer) was performed in the presence of 5 mM phenylmethanesulfonyl fluoride for 8 h at 25 oe. The amount of enzyme required for complete digestion was previously determined by serial dilution.

Electrophoresis and Peptide Mapping

Sampies were prepared for electrophoresis by dialysis against 1 mM ammonium bicarbonate followed by Iyophilization. Dry sampies were dissolved in 50,u1 of electrophoresis sampie buffer which contained 7.7 mg/mi of dithiothreitol, and denatured by heating at 100°C for three minutes. Labeled medium proteins from one dish of cells (250,000 cells per dish) were applied to each slot. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed by the method of Laemmli (1970) using 5 0/0 vertical si ab gels measuring 15 cm X 15 cm X 1.3 mm. The buffers and gels included 2 M urea to obtain optimal separation of pro a1(I) from pro a1(III). Immediately after electrophoresis, gel strips corresponding to individual lanes, measuring approximately 10 cm X 0.5 cm, were cut out of the wet gel and rinsed with 15 ml of 70 Ofo formic acid (viv). In some experi­ments, gel strips were first treated with 5 ml of 5 0/0 mercaptoethanol (viv) for 30 min at 70 oe. After three successive 30 min rinses with 70 % formic acid, each gel strip was incubated in 5 ml of 70 Ofo formic acid which contained 50 mg/ ml of CNBr, and gently agitated for 12 h at 25 oe. The digestion was terminated by successive rinses with a buffer solution which contained 100 mM Tris, pH 6.8, 30 Ofo glycerol (viv), and bromophenol blue. In experiments designed to determine

Peptide Mapping of Collagen Chains Using CNBr Cleavage 545

iE soluble radioactivity was released from gel strips, 1 ml aliquots of each rinse (the formic acid, CNBr, and Tris buffer solutions) were assayed for radioactivity in a liquid scintillation counter.

Each gel strip was rinsed with the Tris buffer solution until the formic acid was neutralized (as indicated by the bromophenol blue indicator dye), then placed horizontally over a second slab gel which contained a 12.5 °/0 seperating gel and a 5 °/0 stacking gel. The second gel was prepared at a width oE 1.6 mm, so that the "first dimension" gel strips could be easily positioned directly over the "second dimension" stacking gel with the aid of a spatula. A 100 mM Tris buffer, pH 6.8 which contained 5 Ofo SDS (w/v), 20 Ofo glycerol (viv), and bromophenol blue was then layered over the first dimension gel strips to a height of 1 cm, and the CNBr-derived peptides were separated by electrophore~is at a current of 20 milliamps. Gels were processed for fluorography with EN3HANCER (New England Nuclear). Assignments for peptides derived from a chains were based on comparisons with the mobilities of known standards (Piez, 1976). Assignments for peptides derived from pro a and TCA chains were then inferred by compari­son with the a chain peptide map.

Results

Evaluation 01 Procedures

After electrophoresis in the second dimension, fluorographs showed no residual radioactivity in first dimension gel strips (data not shown). Furthermore, aliquots of the formic acid, CNBr, and Tris buffer solutions used for incubation of the first dimension gel strips contained no detectable radioactivity (data not shown). This suggested that diffusion of collagen chains and their CNBr-derived peptides trapped in a 5 Ofo polyacrylamide gel was minimal under the conditions used here, and that these peptides were recovered quantitatively by electroelution in the presence of 5 Ofo SDS (w/v).

Peptide maps are shown in Figure 1. Cleavage of pro a (Figure 1 A), a (Figure 1 B), and TC"" (Figure 1 C) chains was incomplete, but the extent of cleavage is comparable to that seen when isolated chains are digested with CNBr (Bornstein and Sage, 1980).

Pretreatment with mercaptoethanol and/or increasing the concentration of CNBr 10-fold did not improve the extent of cleavage which suggests that the uncleavaged methionyl residues are either oxidized to sulfones or are sterically "protected" from CNBr.

Comparison 01 Peptide Maps

Pro a chain peptide maps (Figure 1 A) show that pro a l(III), pro a 1(1), and pro a 2(1) are easily identified by their CNBr-derived fragments. In addition, partially processed pro a 1(1) and pro a 2(1) chains are evident in Figure 1 A, tentatively identified as pC a 1(1) and pC a 2(1), since these bands are only present on reducing gels (data not shown).

There an; several differences between the pro a and a chain peptide maps (Figures 1 A and 1 B, respectively), especially in the partial cleavage peptide regions, which suggests that additionally protected methionyl residues or pre-

546 G. S. Barsh, K. E. Peterson and P. H. Byers

Fig. 1. Peptide maps of pro a chains (A) , a chains (B), and TCA chains (C). Proteins were labeled and fractionated as described in Materials and Methods. Replicate first dimension fluoro grams are displayed horizontally and second dimension fluorograms are displayed vertically. Each peptide map represents the medium proteins from 250,000 cells labeled during a 16-hour incubation. First dimension fluorograms are exposed for 1 day; second dimension fluoro grams are exposed for 3-5 days. al(I)CB3 is apparent on longer ex­posures of CNBr-cleaved a chains and TCA chains. The identity of pNa2(I) is inferred from its mobility and CNBr-derived fragments.

cursor-specific extensions are present on these pro a chain-derived fragments. In particular, the size of the a 2(I)CB 3-5 peptide in the a chain peptide map is 3000-5000 daltons smaller than the corresponding peptide from pro a 2(1) or pC a 2(1), which suggests that the first accessible methionyl residue in the pro a 2(1) carboxyl extension is at least 40-50 residues into the C-propeptide.

By comparing the a chain to the TCA chain peptide map, we were able to identify peptides which contained the mammalian collagenase cleavage site; these are identified as a 2(1) eB 3-SA and a 1(1) CB 7A in Figure 1 C.

Discussion

We have described a new peptide mapping technique using CNBr cleavage of collagen chains trapped within polyacrylamide gels. The two dimensional format allows all the collagens produced by as few as 250,000 cdls to be examined in a single array without prior chromatographie or enzymatic purification, and facilitates the comparison of relative mobilities from one map to another.

Peptide Mapping of Collagen Chains Using CNBr Cleavage 547

A common problem encountered with the analysis of proteins within gels is recovery of the separated molecules for subsequent chemical and enzymatic procedures (Bray and Brownlee, 1973; Brown and Howard, 1980). In some cases, we have recovered as little as 10 ng of pro tein by including high concentrations of SDS in the electroelution buffer. In our hands, diffusion of peptides from the gel during the digestion procedure was negligible.

Two other peptide mapping techniques used to identify collagens are enzymatic digestion of proteins within gel slices (Cleveland et al., 1977; Robey and Martin, 1981), and a recently described two dimensional technique using proteinase K digestion of proteins radioiodinated within gel slices (Sage et al., 1981). The method described here provides a third way to analyze ehe structure of a collagen chain without prior isolation, and in addition, is weil suited for structural studies of chains produced by cells in sm all quantities. For ex am pie, genetic variants with altered electrophoretic mobilities have been localized to a particular region within their parent collagen chains (Barsh and Byers, 1981; Byers et al., in press).

In this study we used radioactive proline to probe the structure of the helical domains of collagenous proteins. However, radioactive cysteine or mannose could be easily substituted, to examine the structures of the propeptide domains and the carbohydrate attachment regions, respectively. Since only a small number of cells are needed, this technique may be especially valuable for analysis of colla­gens produced by primary cultures which cannot be passaged in vitro, such as certain glomerular epithelial cells (Killen and Striker, 1979), or chondrocytes which exhibit an unstable phenotype (Benya et al., 1978). Finally, this method should aid in the rapid identification of new collagen types produced by cells in culture (Sage et al., 1980) as our knowledge of the collagen gene family continues to expand.

Nomenclature. The system of Bornstein and Sage (1980) is used for referring to pro a chains, a chains, and their CNBr peptides. TCA refers to the amino terminal fragment of collagen after c1eavage with mammalian collagen ase; a1(I)A and a2(I)A, respectively refer to the maino terminal portion of a1(I) and a2(I) after c1eavage with mammalian colla­genase; similarly, a2(I)CB3-5A and a1(I)CB7A, respectively refer to the amino terminal portion of a2(I)CB3-S and a1(I)CB7 after c1eavage with mammalian collagenase.

Acknowledgments

Supported in parts by grants AM 21557, AM 21897 and GM 07266 from the National Institutes of Health, a Clinical Research grant from the March of Dimes Birth Defects Foundation, and a supply grant from the Poncin Fund. Gregory S. Barsh is a predoctoral trainee of the Medical Scientist Training Program at the University of Washington (GM 07266); Peter H. Byers is an Established Investigator of the American Heart Association.

References

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Benya, P., Padilla, S. R. and Nimni, M. E.: Independent regulation of collagen types by

548 G. S. Barsh, K. E. Peterson and P. H. Byers

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Dr. Peter H. Byers, Department of Pathology SM-30, University of Washington, Seattle, WA 98195, USA.