The Distribution and Initial Characterization Of

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”/electrophoresis. 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 subsequent 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 propeptides 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. The migration positions of internal collagen buffer (pH 6.5) (12) and digestion with endo H was performed in 500 standards are shown at the top of each panel. The migration positions ~1 of 0.1 M sodium citrate buffer (pH 5.5) (13). Digestions were of alA and a2* are 42 mm and 47 mm, respectively. Total length of continued at 37°C for 24 h using 10 milliunits of enzyme and a drop the gel was 100 mm. A, unreduced; B, reduced. M, [“‘Clproline- of toluene. Reactions were stopped by freezing the sample. To labeled material; 0- - -0, [3H]glucosamine-labeled material. monitor the release of oligosaccharide units, samples were chroma- tographed on P-6 as described above. Recovery of radioactivity was glucosamine was subjected to digestion by partially purified essentially quantitative. human skin collagenase and the products were monitored by Alkaline-borohydride treatment of glycopeptides was performed as SDS/electrophoresis before and after disulfide bond reduc- described by Zinn et al. (14). Samples were hydrolyzed in 500 ~1 of tion (Fig. 1). The migration positions of pro-yB (unreduced 1 M NaOH containing 4 M NaBH4 at 80°C for 24 h. After cooling and addition of 500 ~1 of distilled water, the sample was adjusted to sample) and pro-al*, pro-cy2*, pro-alB, and pro-cy2B (reduced pH 5 by dropwise addition of glacial acetic acid. After centrifugation samples) were established using [‘%]proline-labeled material to remove insoluble material, the supernatant was applied directly to (Fig. 1, solid Zine).5It can be seen that with the [“Hlglucosa- a P-6 column as described above. Recovery of radioactivity was mine-labeled sample (Fig. 1, dashed line), essentially all of essentially quantitative. the radioactivity was associated with the COOH-terminal Molecular weights of glycopeptides and oligosaccharides were es- fragments. Subsequent digestion of human skin collagenase- timated using the calibration curve of Etchison et al. (11). To ascer- derived fragments with bacterial collagenase (data not shown) tain the validity of this, a glycopeptide of known molecular weight (Mr = 3600)4 and bacitracin (MT = 1423) were both tested and found gave gel filtration patterns virtually identical with those ob- to elute in the predicted volume. tained by bacterial collagenase digestion alone (2). Distribution of Carbohydrate betweenpro-al andpro-&?-- RESULTS Native procollagen labeled with either [ ‘%]proline, [“HIman- Distribution of Carbohydrate between the COOH- and nose, or [“Hlglucosamine was subjected to SDS/electropho- NH&erminal Propeptides of Extracellular Procollagen- resis under reducing conditions. The migration positions of Native procollagen labeled with either [‘%]proline or [“HI- pro-al and pro-a2 were established by using [‘%]proline- ’ The abbreviations used are: SDS, sodium dodecyl sulfate; Con A, ’ The nomenclature of animal collagenase-derived fragments from concanavalin A, WGA, wheat germ agglutinin; endo D, endo-P-N- procollagen has been established (16): pro-ya represents the disulfide- acetylglucosaminidase D; endo H; endo-P-N-acetylglucosaminidase bonded COOH-terminal fragment which yields pro-alB and pro-a2B H. upon reduction; pro-al* and pro-a2* represent the NH*-terminal 4 Clayton A. Buck, personal communication (15). fragments. 10800 Oligosaccharide Units on Type I Procollagen

tography of reduced and alkylated procollagen labeled with either [“Hlglucosamine (Fig. 4) or [“Hlmannose (Fig. 5). The identity of Pool I as pro-a2 and Pool II as pro-al was verified by SDS/electrophoresis (Figs. 2B and 3B). Distribution of Carbohydrate between the Propeptides of Pro-al and Pro-&-Reduced and alkylated pro-al and pro- cu2 chains prepared by DEAE-cellulose chromatography and labeled with either [“Hlglucosamine or [3H]mannose were digested with purified bacterial collagenase in order to generate the NH,- and COOH-terminal propeptides from each chain. These were then resolved by SDS/agarose gel filtration on A-1.5m (data not shown). The results were similar to those observed previously (2) in that essentially none of the [“HI- glucosamine or [“Hlmannose was associated with the NH2- terminal propeptides. A similar set of experiments was performed with [‘Hltyro- sine-labeled procollagen in order to prepare labeled NHZ- terminal propeptides as well as COOH-terminal propeptides from pro-c*1 and pro-a2 (data not shown). The propeptide samples were subjected to affinity chromatography on lectin- MIGRATIONMlGR~kN (mm) MIGRATION (mm) linked agarose as described under “Methods.” The results are FIG. 2 (left). Electrophoretic profile on SDS/electrophoresis presented in Table I. It can be seen that the NHs-terminal of: A, reduced [3H]glucosamine-labeled procollagen purified propeptides were not specifically bound to either Con A Downloaded from by DEAE-cellulose chromatography (2); and B, reduced and (specific for a-D-&COSe and a-n-mannose) or WGA (specific alkylated procollagen fractions purified by DEAE-cellulose for N-SCetyl-D-glUCOSSmine). On the other hand, the COOH- chromatography. w, Pool II from Fig. 4; 0- - -0, Pool I from terminal propeptides exhibited a binding to Con A-Sepharose Fig. 4. The migration positions of internal collagen standards are similar to that of ovalbumin (a glycoprotein containing both shown at the top of Panel A. FIG. 3 (right). Electrophoretic profile on SDS/electrophore- mannose and N-acetylglucosamine) in the presence of either sis of: A, reduced [3H]mannose-labeled procollagen purified the nonspecific sugar galactose or the specific sugar analogue www.jbc.org by DEAE-cellulose chromatography (2); and B, reduced and alkylated procollagen fractions purified by DEAE-cellulose chromatography. M, Pool II from Fig. 5; 0- - -0, Pool I from Fig. 5. The migration positions of internal collagen standards are shown at the top of Panel A. by guest, on July 22, 2011

“; xm~xc$j~~j5’ - 0 50 150 zoo0 EFFLUENT’%LUME (ml) FIG. 5. Elution profile of reduced and alkylated [‘HIman- nose-labeled procollagen on DEAE-cellulose. See legend to Fig. 4 for details.

EFFLUENT VOLUME (ml) TABLE I Affinity chromatography of [3HJtyrosine-labeled propeptides 4. Elution profile of reduced and alkylated [3H]gluco- FIG. Per cent &ted samine-labeled procollagen on a column (1.5 x 15 cm) of DEAE-cellulose at room temperature. After application of the Lectin Additions o~;vay- cpsg$. ,p;g. NHa sample, the column was washed initially with 50 ml of starting buffer Pep- peptide peptide tide? (50 mM Tris. HCl, 6 M urea, 1 mM dithioerythritol, 1 mu benzamidine. HCl, 1 mM phenylmethanesulfonyl fluoride, 0.1% Triton X-100, 2 mM % EDTA, pH 8.6) and elution was continued with a 2O@ml linear +0.5 M Galactose 21 31 9 85 gradient of 0 to 0.3 M NaCl in starting buffer. Fractions of 2.4 ml were Con A collected and those indicated by the bars were pooled for subsequent +0.5 M a-Methyl- 100 85 91 85 experiments. M, radioactivity; X-.-X, conductivity. mannoside

None 29 82 pro-al/pro-a2 labeled material, and the ratio of ranged from WGA n.d.’ nd. 1.5 to 1.8 under these conditions (see Fig. 4A of Ref. 1). +0.4 M N-Acetyl- 99 100 However, the ratio of pro-al/pro-a2 for [3H]glucosamine-la- glucosamine beled procollagen ranged from 1.2 to 1.6 (Fig. 2A) while that a Recovery was determined by absorbance measurements at 280 for [“Hlmannose-labeled procollagen ranged from 0.8 to 1.2 nm. (Fig. 3A). ’ Includes propeptides from pro-01 and pro-n2. Similar results were obtained by DEAE-cellulose chroma- ’ n.d., not determined. Oligosaccharide Units on Type I Procollagen 10801

4- 4-

2- 2-

o- o- q 4. 4-

2- 2-

VO 4 4 “s’ 40 60 60 20 O2c 40 60 80 _hFRACTION NUMBER FRACTION NUMBER FRACTION NUMBER FIG. 6 (left). Elution profile on a polyacrylamide gel (Bio-Gel cosidase digestion or alkaline-borohydride hydrolysis.

P-6, 200 to 400 mesh) column (1.5 x 130 cm) of glycopeptides FIG. 7 (center). Elution profile on P-6 of the products of Downloaded from derived from pronase digestion of [3H]mannose-laheled endoglycosidase digestion of pronase-derived [3H]man- COOH-terminal propeptides. A, pro-a2; B, pro-oil. Before appli- nose-labeled glycopeptides. A, pro-al; B, pro-&. M, endo H; cation of the sample, ovalbumin and “Hz0 were added to mark the 0- - -0, endo D. See legend to Fig. 6 for details. excluded volume ( VO) and the totally included volume (V,), respec- FIG. 8 (right). Elution profile on P-6 of the products of alka- tively. The elution position of a glycopeptide standard (Mr = 3600) line-horohydride hydrolysis of pronase-derived [3H]mannose- is indicated by the unmarked arrow. The column was eluted with 0.1 labeled glycopeptides. A, pro-al; B, pro-u2. For reference, the M ammonium acetate (pH 6.0) and 2%ml fractions were collected. elution position of the original glycopeptide (G) is indicated (see Fig. www.jbc.org Samples were pooled as indicated by the bar and used for endogly- 6). See legend to Fig. 6 for details. cY-methyhnannoside. At this time, the significance of the ap- >90% of the glucosamine and 100% of the mannose was parent difference in binding specificity between pro-al and associated with the COOH-terminal propeptide (2). The ab- pro-a2 COOH-terminal propeptides is not known. sence of mannose in the NHz-propeptide has recently been by guest, on July 22, 2011 Partial Characterization of Glycopeptides from the corroborated by two other laboratories (4, 17). To further COOH-terminal Propeptides of Pro-al and Pro-(u2-[3H]- investigate the previous observation that a small amount of Mannose-labeled COOH-terminal propeptides isolated by [3H]glucosamine may be present in the NH*-terminal propep- SDS/agarose gel fitration were digested with pronase as tide of type I procollagen (2), we performed a series of exper- described under “Methods.” The results of gel filtration of the iments employing mammalian collagenase digestion of intact digest are shown in Fig. 6. A single peak of radioactivity was procollagen, and affinity chromatography and bacterial col- detected for both pro-a2 (Fig. 6A) and pro-al (Fig. 6B). Using lagenase digestion of reduced and alkylated pro-e chains. a molecular weight calibration curve (ll), it was estimated Cleavage of [“Hlglucosamine-labeled procollagen with hu- that the glycopeptides derived from the COOH-terminal pro- man skin collagenase clearly showed that the NHa-terminal peptides of pro-al and pro-a2 chains each had a relative M, fragments, pro-al* and pro-a2*, were not labeled (Fig. 1). = 2000 to 2200. Subsequent bacterial collagenase digestion of these fragments These glycopeptides were subsequently incubated with en- or digestion of isolated pro-ail and pro-a2 chains (data not doglycosidases. As shown in Fig. 7, the glycopeptides from shown) also showed no label associated with the NHa-terminal both pro-n1 and pro-a2 were resistant to endo D and suscep- propeptides. In addition, affinity chromatography on both tible to endo H digestion. The cleaved samples each yielded Con A-Sepharose and WGA-agarose showed that the NHz- a major peak of radioactivity with a relative M, = 1400 to 1500 terminal propeptides of type I procollagen do not contain which presumably represents the entire oligosaccharide unit oligosaccharide units. In contrast, it has been shown recently minus the single N-acetylglucosamine that remains bound to that both the COOH- and NH&erminal propeptides of type the peptidyl asparagine. This was confirmed in another set of II procollagen contain mannose (17). experiments in which the glycopeptides were treated with We also investigated the distribution of carbohydrate be- alkaline borohydride in order to remove completely the pep- tween pro-cl and pro-a2 by both SDS/electrophoresis (Figs. tide moiety and generate the reduced oligosaccharide unit. As 2A and 3A) and DEAE-cellulose chromatography (Figs. 4 and shown in Fig. 8, the hydrolyzed samples each yielded a major 5). In both instances, there appeared to be more mannose per peak of radioactivity with a relative &Z, = 1600 to 1700. chain in pro-a2 than in pro-al. In [“Hlmannose-labeled procollagen, the ratio of pro-al/pro-n2 which we calculated as 0.8 DISCUSSION to I.2 is similar to the value of 1.4 calculated from the Our previous studies showed that freshly isolated matrix- quantitative carbohydrate data of Olsen et al. (3). The signif- free chick embryo tendon cells, when incubated in the pres- icance of pro-a2 having more mannose is not yet clear, but ence of either [2-3H]mannose or [6-3H]glucosamine, synthe- this may be the reason for the apparent difference in binding sized type I procollagen which contained these sugars exclu- to Con A-Sepharose (Table I). sively in the propeptide domain (1). In addition, based upon The results of pronase digestion (Fig. 6) showed that the experiments using bacterial collagenase digestion to generate COOH-terminal propeptides of both pro-al and pro-n2 each the NHZ- and COOH-terminal propeptides, it was shown that contain a glycopeptide unit with an apparent M, = 2000 to 10802 Oligosaccharide Units on Type I Procollagen

2200. In order to separate the peptide moiety from the oligo- to Drs. Roselyn Eisenberg and Gary Cohen for the gift of the glyco- saccharide moiety, the glycopeptides were hydrolyzed in al- peptide standard, and to Joyce S. Knoll and Jacqueline Zavodnick kaline borohydride (14). The results of this cleavage yielded for expert technical assistance. oligosaccharide units with an apparent M, = 1600 to 1700 (Fig. REFERENCES 8). This suggests that each unit probably contains eight to 1. Clark, C. C., and Kefalides, N. A. (1976) Proc. Nutl. Acad. Sci. U. nine sugars. A similar conclusion was reached by Duksin and S. A. 73, 34-38 Bornstein (4). Using tunicamycin, they reported a molecular 2. Clark, C. C., and Kefalides, N. A. (1978) J. Biol. Chem. 253, 47- weight difference of about 2000 between nonglycosylated 51 COOH-terminal propeptides and those from normal procol- 3. Olsen, B. R., Guzman, N. A., Engel, J., Condit, C., and Aase, S. lagen. Collectively, these results suggest that there may be (1977) Biochemistry 16,3030-3036 only a single oligosaccharide unit on each COOH-terminal 4. Duksin, D., and Bornstein, P. (1977) J. Biol. Chem. 252, 955-962 propeptide. 5. Anttinen, H.. Oikarinen, A.. Rvhanen. L.. and Kivirikko. K. I. (1978) hEI& Lett. 87,‘222-22% In order to characterize further the oligosaccharide units, 6. Miyoshi, M., and Rosenbloom, J. (1974) Connect. Tissue Res. 2, the pronase-derived glycopeptides were incubated with either 77-84 endo D or endo H. Only the latter was capable of cleaving 7. Clark, C. C. (1976) in The Methodology of Connective Tissue these structures (Fig. 7). From these results and the known Research (Hall, D. A., ed) pp. 205-226, Joynson-Bruvvers Ltd., specificities of the endogylcosidases, it is possible to speculate Oxford upon the structure of the oligosaccharide units. Both enzymes 8. Blakesley, R. W., and Boezi, J. A. (1977) Anal. Biochem. 82,580- 582 have been shown to cleave between the N-acetylglucosamine 9. Monson, J. M., Click, E. M., and Bornstein, P. (1975) Biochem- residues of the di-N-acetylchitobiose core of asparagine-linked istry 14,4088-4092 oligosaccharide units (18, 19). However, the specificities are 10. Schwarz, R. T., Schmidt, M. F. G., Anwer, U., and Klenk, H.-D. significantly different with respect to the structure of the (1977) J. Virol. 23, 217-226 peripheral sugars. Neutral high mannose type oligosaccha- 11. Etchison, J. R., Robertson, J. S., and Summers, D. F. (1977) Downloaded from rides are susceptible to endo H, but acidic “complex” type Virology 78,375-392 12. Tai, T., Yamashita, K., Ogata-Arakawa, M., Koide, N., Mura- units are resistant (20). This specificity virtually rules out the matsu, T., Iwashita, S., Inoue, Y., and Kobata, A. (1975) J. presence in the procollagen units of terminal sialic acid resi- Biol. Chem. 250, 8569-8575 dues. In addition, it has been shown that fucose linked to the 13. Robbins, P. W., Hubbard, S. C., Turco, S. J., and Wirth, D. F. proximal N-acetylglucosamine of the di-N-acetylchitobiose (1977) Cell 12,893-900 core would completely impair endo H activity (21). Further- 14. Zinn, A. B., Marshall, J. S., and Carlson, D. M. (1978) J. Biol. www.jbc.org more, resistance to endo D suggests that the oligosaccharide Chem. 253,6761-6767 15. Warren, L., Fuhrer, J. P., Buck, C. A., and Walborg, E. F., Jr. contains no unsubstituted cwl, 3-linked mannosyl residues (12). (1974) Miami Winter Symp. 8, 1-16 Thus, the oligosaccharide unit(s) on the COOH-terminal pro- 16. Byers, P. H., Click, E. M., Harper, E., and Bornstein, P. (1975)

peptides of type I procollagen probably consist primarily of Proc. Natl. Acad. Sci. U. S. A. 72, 3009-3013 by guest, on July 22, 2011 N-acetylglucosamine and mannose residues. Future experi- 17. Guzman, N. A., Graves, P. N., and Prockop, D. J. (1978) Biochem. ments will permit determination of the precise structure of Biophys. Res. Commun. 84,691-698 these units. 18. Muramatsu, T. (1971) J. Biol. Chem. 246, 5535-5537 19. Tarentino, A. L., and Maley, F. (1974) J. Biol. Chem. 249, 811- 817 Acknowledgments-I am indebted to Dr. Nicholas A. Kefalides for 20. Tarentino, A. L., Plummer, T. H., Jr., and Maley, F. (1974) J. his advice and generous support, to Drs. Eugene Bauer and John Biol. Chem. 249,818-824 Jeffrey for the gift of human skin collagenase, to Drs. Joel Rosen- 21. Tarentino, A. L., and Maley, F. (1975) Biochem. Biophys. Res. bloom and Paul Christner for the gift of purified bacterial collagenase, Commun. 67,455-462