THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 254, No. 21, Issue of November 10, pp. 10798-10802, 1979 Printed in U.S.A. The Distribution and Initial Characterization of Oligosaccharide Units on the COOH-Terminal Propeptide Extensions of the Pro-al and Pro-a2 Chains of Type I Procollagen* (Received for publication, March 12, 1979, and in revised form, May 25, 1979) Charles C. Clark+ With the technical assistance of Linda Graham From the Connective Tissue Research Institute, University City Science Center, and the Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 A number of experiments were performed to localize Duksin and Bornstein (4) further suggested that the majority the oligosaccharide units on type I procollagen secreted of this carbohydrate was located on the COOH-terminal pro- by matrix-free chick embryo tendon cells. The results peptide( In this study, we present additional evidence which of mammalian collagenase digestion of [3H]glucosa- is consistent with the conclusion that very little (if any) mine-labeled procollagen, lectin affinity chromatogra- carbohydrate is associated with the NHz-terminal propeptides phy of [3H]tyrosine-labeled propeptides, and bacterial of either pro-al (type I) or pro-a2. The results also suggest collagenase digestion of [3H]glucosamine- or [3H]man- that each COOH-terminal propeptide in extracellular type I Downloaded from nose-labeled pro-al and pro-a2 chains indicated that procollagen contains a single “high mannose”-type oligosac- the COOH-terminal propeptides contain the majority charide unit consisting of approximately eight to nine mono- (if not all) of the oligosaccharide units in the propeptide saccharides. domains. Analysis of sugar-labeled pro-al and pro-a2 EXPERIMENTAL PROCEDURES chains by sodium dodecyl sulfate-polyacrylamide gel MateriaZs-L-[ring-3,5-3H]tyrosine was purchased from New Eng- electrophoresis and DEAE-cellulose chromatography land Nuclear; diethylaminoethyl (DEAE) cellulose (DE-52) from suggested that the pro-a2 chain may contain more man- Whatman; concanavalin A (Con A)-Sepharose from Pharmacia; www.jbc.org nose than the pro-al chain. Subsequent pronase diges- wheat germ agglutinin (WGA)-agarose from E-Y Laboratories; apro- tion of isolated [3H]mannose-labeled COOH-terminal tinin, D-galactose, N-acetyl-n-glucosamine, a-methyl-n-mannoside, propeptides from purified pro-al and pro-a2 yielded bacitracin, bovine serum albumin, and Coomassie Brilliant Blue G only a single glycopeptide fraction from each with an from Sigma; and pronase from Calbiochem-Behring. Bacterial colla- approximate M, = 2000 to 2200. These glycopeptides genase from Worthington (CLSPA) which was purified by isoelectric by guest, on July 22, 2011 were resistant to cleavage by endo+-N-acetylglucosa- focusing (6) was kindly donated by Drs. Joel Rosenbloom and Paul minidase D, but were susceptible to endo+N-acetyl- Christner (University of Pennsylvania); and partially purified human skin collagenase was kindly provided by Drs. Eugene A. Bauer and glucosaminidase H yielding oligosaccharide units of John J. Jeffrey (Washington University). Endo-P-N-acetylglucosa- approximate Mr = 1400 to 1500. Alkaline-borohydride minidase D (endo D) from Diplococcuspneumoniae (lot No. ED8301) hydrolysis of the pronase-derived glycopeptides and endo-P-N-acetylglucosaminidase H (endo H) from Streptomyces yielded oligosaccharide units of approximate M, = 1600 griseus (lot No. EH6XO4) were obtained from Miles Laboratories and to 1700 from each pro-a chain. used without further purification.’ The source of all other materials Taken collectively, these results indicate that each has been reported previously (1, 2). COOH-terminal propeptide in type I procollagen con- Methods-Extracellular native procollagen labeled with either L- tains a single type of ‘%igh mannose” oligosaccharide [U-‘4C]proline, L-[ring-3,5-“Hltyrosine, n-[2-3H]mannose, or D- unit consisting of approximately eight to nine mono- [6-‘Hlglucosamine was prepared and purified by ammonium sulfate precipitation and DEAE-cellulose chromatography as described pre- saccharides. viously (2) except that aprotinin (5 trypsin inhibitor units) was included in the incubation mixture. Previous analyses (1, 2) have shown that the majority of the labeled carbohydrate precursor ac- tually appeared in procollagen as either glucosamine or mannose, Recent results from both om laboratory (1, 2) and those of respectively. others (3-5) have collectively shown that the propeptide do- Human skin collagenase digestions of native procollagen were mains of type I procollagen contain asparagine-linked oligo- performed in 1 ml of 0.4 M NaCl, 0.1 M Tris. HCl (pH 7.4) containing saccharide unit(s) whose composition includes N-acetylglu- 5 rnM CaC12, and 50 pl* of enzyme solution. Incubation was continued cosamine and mannose. Our findings (2) as well as those of for 3 h at 27°C and the reaction was stopped by addition of EDTA to 10 mM final concentration. After dialysis against 0.01 M pyridine, samples were lyophilized. * This investigation was supported by National Institutes of Health Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was Grants AM-20553 and HL-15061. A preliminary report of this work performed as described previously (1, 7) except that Coomassie Bril- was presented at the annual meeting of the Society for Complex liant Blue G-250 was used as a stain (8). The polymerizing solution Carbohydrates held in Washington, D. C., September 27 to 29, 1978. contained 7% acrylamide monomer and 0.137% N,N’-diallyltartar- The costs of publication of this article were defrayed in part by the diamide as a cross-linking agent. After staining, each gel was fraction- payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 ’ As stated by the manufacturer, endo D preparations contained solely to indicate this fact. no activities of (Y- and ,&galactosidase, (Y- and /3-mannosidase, a-~- $ Recipient of National Institutes of Health Research Career De- fucosidase, or proteases. Occasionally, a trace amount (-1%) of ,B-N- velopment Award 5 K04 AM-00360. To whom requests for reprints acetylglucosaminidase was detected. Endo H preparations contained should be addressed at the Connective Tissue Research Institute, no detectable activity of various exoglycosidases or proteases. University City Science Center, 3624 Market St., Philadelphia, Pa. * E. A. Bauer, personal communication. Enzyme (50 to 100 ~1) will 19104. digest 100 to 200 fig of collagen in 30 to 60 min at 27°C. 10798 Oligosaccharide Units on Type I Procollagen 10799 ated into l-mm slices and each slice was solubilized and counted as described previously (1). In order to separate pro-al and pro-a2 chains, native procollagen was reduced and alkylated essentially as described by Monson et al. (9). Samples were then dialyzed in the dark at room temperature against 6 M urea containing 50 IIIM Tris.HCl (pH 8.6 at 22”C), 1 mM dithioerythritol, and 0.1% Triton X-100, with 1 mM benzamidine. HCl, 1 mM phenylmethanesulfonyl fluoride, and 2 mM EDTA as protease inhibitors. Subsequent chromatography was performed at room temperature on DEAE-cellulose. After elution with 50 ml of the above buffer, the sample was eluted using a linear gradient from 0 to 0.3 M NaCl over a total volume of 200 ml. Recoveries of radioactivity ranged from 60 to 85%. Appropriate fractions were pooled, dialyzed against 0.01 M pyridine at 4”C, and lyophilized. The identity of material in the pooled fractions was assessed by SDS”/electrophore- sis. To generate the NH,- and COOH-terminal propeptides, samples oi reduced and alkylated pro-al and pro-a2 chains were each digested with purified bacterial collagenase and the propeptides were sepa- rated by gel ftitration on SDS/agarose (2). Appropriate fractions were pooled, dialyzed against 0.01 M pyridine, and lyophilized for subse- quent experiments. Affinity chromatography was performed at room temperature on 3-ml columns of lectin-bound agarose. Con A-Sepharose columns were equilibrated in 0.1 M NaCl, 25 mu Tris. HCl (pH 7.5) containing 1 mM CaCL, 1 InM MgC12, 1 mM MnCL, 0.1 mg/ml of bovine serum albumin, 0.1% Triton X-100,0.2% SDS, 0.01% sodium azide, and either Downloaded from 0.5 M galactose or 0.5 M or-methylmannoside. WGA-agarose columns were equilibrated in 0.1 M NaCl, 25 InM Tris. HCl (pH 7.5) containing 0.1 mg/ml of bovine serum albumin, 0.1% Triton X-100, and 0.01% sodium azide with or without 0.4 M N-acetylglucosamine. Samples isolated from SDS/agarose and containing 5,000 to 10,COO cpm were applied to these columns in 0.5 ml of the appropriate buffer and eluted with 10 ml of the same buffer. One-milliliter fractions were www.jbc.org collected and counted. Ovalbumin was used as a control. Pronase digestion of reduced and alkylated COOH-terminal pro- peptides from pro-al and pro-a2 chains was carried out in 500 ~1 of 0.1 M Tris. HCl (pH 8.0) containing 2 ITIM CaC12. To each sample was added 3 mg of predigested (10) enzyme and a drop of toluene to by guest, on July 22, 2011 prevent microbial growth. The digestion was continued for 100 h at 37°C with 3-mg portions of pronase added every 24 h. The reaction was stopped by the addition of EDTA to 10 mu final concentration 20 40 60 and the digest was immediately analyzed by gel fdtration on a column MIGRATION (mm) (1.5 x 130 cm) of P-6 equilibrated and eluted with 0.1 M ammonium acetate (pH 6) containing 0.01% sodium azide (11). Recovery of FIG. 1. Electrophoretic profile on SDS/electrophoresis of radioactivity was essentially quantitative after this procedure. fragments derived by human skin collagenase digestion of Digestion with endo D was performed in 500 pl of 0.15 M phosphate native procollagen.