The Structure of Avian Type XI1 Collagen - The Journal of Biological ...

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Aug 5, 2016 - Mayne, University of Alabama in Birmingham) (upper row) and with the 75d7 ..... nochemistry of the Extracellular Matrix (Furthmayr, H., ed) pp.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 22, Issue of August 5, pp. 13150-13156.1989

Printed in U.S.A.

The Structureof Avian Type XI1 Collagen al(XI1) CHAINS CONTAIN 190-kDa NON-TRIPLE HELICAL AMINO-TERMINAL DOMAINS AND FORM HOMOTRIMERIC MOLECULES* (Received for publication, March 9, 1989)

Bernard Dublet$, Suk Oh$, Stephen P. SugrueQ, MarionK. Gordon$, Donald R. Gereckeg, Bjdrn R. OlsenB,and Michel van derRest$ll From the $Genetics Unit, Shriners Hospital, Montreal, QC, Canada H3G lA6 and the $Department of Anatomy and CeUulur Bwbgy, Harvard Medical School, Boston,Massachusetts 02115

The monoclonal antibody 75d7, specific for type XI1 different from the other matrix constituents described collagen (Sugrue, S.P., Gordon, M. K., Seyer, J., Dub- so far. let, B., van der Rest, M., and Olsen, B. R. (1989) J . Cell Biol., in press), was used to characterize the intact form of type XI1 collagen from chick embryo leg tenThe organization of extracellular matrices is the result of a dons. On an immunoblot of a 6% polyacrylamide gel of tendon extracts, one sharp bandis recognized by the precise spatial arrangement of a number of structural macantibody at M,= 220,000, while twofuzzy and poorly romolecules. Among these, the fibrillar collagens play a promresolved bands are seen at M , = 270,000 and M , = inent role as they provide tensile strength to tissues. The 290,000. By immunoprecipitation of radiolabeled ten- major fibrillar collagen in most tissues is type 1, while in don culture media and electrophoresis of the precipi- hyaline cartilage and some other tissues, such as thevitreous tated material, bands with the same mobilities are body, it is type I1 collagen. Additional quantitatively minor observed, indicating that type XI1 collagen is not pro- fibrillar collagens participate in mixed (heterotypic) fibrils teolytically processed in the extracellularspace. Type with the major fibrillar types in some and possibly in all XI1 collagen was extracted from tendons with 1 M NaCl tissues (1-3). Types I11 and V collagens are both associated by concan- with type I in soft connective tissues, while type V has been in a Tris-HC1 buffer and partially purified avalin A-Sepharose and gel permeation chromatogradetected in mineralized type I-containing tissues (1,4).Type phies, using dot immunoblots to monitor the purificaXI collagen has been described in type 11-containing tissues tion. Fractions highly enriched in bacterial collagen(2). One possible function for the minor fibrillar collagens is ase-sensitive proteins with the same electrophoretic properties as type XI1 collagen were obtained. These that theycontribute to theregulation of fibril diameters. In addition to fibrillar collagens, several other collagen fractions did not stain with Alcian blue and neither they nor the immunostained type XI1 collagen were types have been described that cannot form the quarterstaggered fibrils characteristic of fibrillar collagens, as their affected by chondroitinase ABC digestion, indicating that type XI1 collagen is not a proteoglycan. A disul- triple helices are of different sizes and contain imperfections fide-bonded trimeric CNBr peptide was isolated by and interruptions. Several of these collagens, such as types affinity chromatography on an antibody column and IV,VI, and VII, have been shown to be involved in the further purified by gel electrophoresis. Its NHt-ter- formation of distinct histological structures, such as basement minal amino acid sequence was shown to be unique, membranes, beaded filaments, and anchoring fibrils, respecdemonstrating that type XI1 collagen is a homotrimer tively (5-7). In contrast,type IX collagen, also unable to form [al(XII)ls. After bacterial collagenase digestion, both quarter-staggered fibrils alone, has been shown to interact the immunopurified radiolabeled preparation and the covalently with type I1 collagen fibrils to form what is thought purified tendon extract fraction showed by gel electrophoresis the presence of a large disulfide-bonded, 3 X to be a link between the fibrils and other, yet unidentified, lSO-kDa, collagenase-resistant domain. Rotary shad- matrix components (8-10). Type IX collagen is only expressed in type 11-containing XI1 owing and electron microscopy of the purified type fraction demonstrated that themolecule has the struc- tissues. On the basis of the hypothesis that molecules perture of acrossconsisting of a 75 nm collagenase- forming a similar function are likely to be required also in sensitive tail, a central globule, and three60 nm arms type I-containing tissues, some of us (M. K. G., D. R. G., and a small globule. After heat denaturation B. R. 0.) isolated from a chick embryo tendon cDNA library each ending in and renaturation,only a very largeglobule can be seen, a cDNA clone (pMG377) that encodes a collagen showing a attached to the triple helical tail. These results show region of sequence similarity to type IX collagen (11).This that type XI1 collagen has a unique structure and is collagen chain has been given the name al(XI1). Thisdiscov* This work was supported in part by the Canadian Medical Re- ery wasfollowedby the isolation of triple helical pepsinsearch Council (Grant MT-7796) and theShriners of North America resistant fragments derived from type XI1 collagen, first from (to M.v.d. R.) and by the National Institutes of Health (Grants chick embryo tendons and, more recently, from the bovine AR36819 and AR3620) (to B. R. 0.).The costs of publication of this periodontal ligament (12, 13). article were defrayed in part by the payment of page charges. This A cDNA-derived non-triple helical sequence of the al(XI1) article must therefore be hereby marked “aduertisernent” in accord- chain has been used to synthesize an oligopeptide which ance with 18 U.S.C. Section 1734 solely to indicate this fact. ll Senior scholar from the Fonds de la Recherche en Santi! du served as antigen for the production of a monoclonal antibody Quebec. To whom correspondence and reprint requests should be directed against type XI1 collagen. The characterization of this antibody and immunolocalization of type XI1 collagen addressed.

13150

Structure of Type X I I Collagen

13151

For analysis of aliquots of the chromatography fractions, the proteins were precipitated with 10% trichloroacetic acid for 30 min a t room temperature. After centrifugation (12,000 x g, 15 rnin), the pellet was washed twice with 100%ethanol and resuspended in sample buffer. Immunoblotting of Peptides-After electrophoresis, the gelwas soaked in transferbuffer (10 mM CAPS, pH 11.0, 10% methanol) for 5 min. During this time, an Immobilon' polyvinylidene difluoride (PVDF) transfer membrane (Millipore) was rinsed with 100% methMATERIALS AND METHODS anol and soaked in transfer buffer. The gel, sandwiched between a Collagen Extraction-Leg tendons, dissected from 60 dozen 17-day sheet of PVDF membrane and several sheets of blotting paper (also chick embryos, were extracted for 48 h at 4 "C with a 50 mM Tris- equilibrated in transfer buffer), was assembled into a blotting appaHC1 buffer, pH 8.0, containing 1M NaCl and a mixture of proteinase ratus (Trans-Blot" Cell, Bio-Rad). After electroelution (20 h at 60 V) inhibitors (1 mM phenylmethanesulfonyl fluoride, 1 mM p-amino- in transfer buffer, the PVDF membrane was soaked 3 X 20 min in benzamidine, 10 mM N-ethylmaleimide, and 10 mM EDTA). The phosphate buffer (10 mM NaH2P04,pH 6.8,0.15 M NaC1) containing suspension was centrifuged (12,000 X g, 20 min), and the supernatant 5% low fat milk. The membrane was then successively rinsed (3 X 5 (extract) was kept at -20 "C. min) in phosphatebuffer, incubated 1h with the monoclonal antibody Preparation of Denatured Collagen-The extract was precipitated diluted (1:400) in phosphate buffer, rinsed (3 X 5 min) in phosphate with ammonium sulfate (40% saturation). After 48 h at 4 "C, the buffer, and soaked 10 min in phosphate buffer containing 5% low fat ammonium sulfate precipitate was centrifuged (12,000 x g, 20 min). milk. It was then incubated 1h with peroxidase-conjugated goat antiThe pellet was resuspended in a 50 mM Tris-HC1 buffer at pH 7.2, mouse antibody (Boehringer) diluted (1:lOOO)in the phosphate buffer containing 0.1 M NaC1, 0.2% sodium dodecyl sulfate (SDS)? and 1 containing 5% normal goat serum ( E N ImmunoBiologicals). After mM EDTA. This material was denatured by heating at 80 "C for 5 washing (3 X 5 min) in phosphate buffer, the membrane was treated min. After centrifugation (12,000 x g, 20 rnin), the solubilizedproteins with 3-3'-diaminobenzidine (Sigma) (0.5 mg/ml) in phosphate buffer were chromatographed on a Sephacryl S-500 (Pharmacia LKB Bio- containing 0.03% H202. The reaction was stopped by a tap water technology Inc.) column (2.6 X 90 cm, 13.4ml/h) at room temperature wash. The molecular weight markers were stained with 0.1% Amido with the same buffer. The effluent was monitored at 214 nm, and 8- Black in methanol/acetic acid/water (45:7:48) for 10-15 min. The ml fractions were collected, dialyzed against 0.5 M acetic acid, and blots were then destained in methanol/acetic acid/water (45:7:48) for stored a t -20 "C. 5-10 min. Preparation of Nondenatured Collagen-The extract was diluted Fibroblust Culture and Biosynthetic Labeling-Tendon fibroblasts by adding an equal volume of a solution of 50 mM Tris-HC1, adjusted were isolated from 17-day-old chick embryos as described (15). The to pH 7.4, and chromatographed on a ConA-Sepharose (Pharmacia) cells were incubated in suspension in Krebs 11-glucose at a concencolumn (2.6 X 12 cm, 4 ml/h) at room temperature. The column was tration of 7.5 X lo6 cells/ml for 7 h a t 37 "C in the presence of 17 pCi washed with a solution of 50 mM Tris-HC1, pH 7.4, containing 0.5 M of [3H]proline/ml. After incubation, the cells were pelleted by cenNaC1, until the absorbance a t 214 nm reached zero and then eluted trifugation in a table-topcentrifuge. Protease inhibitors (1mM phenwith 1 M methyl a-D-glucopyranoside dissolved in the same buffer. ylmethanesulfonyl fluoride, 1 mM p-aminobenzamidine, 10 mM NFractions (2 ml) were collected and analyzed by gel electrophoresis. ethylmaleimide, and 10 mM EDTA) were added tothe medium. The fractions containing type XI1 collagen were pooledand chromat- Medium proteins were precipitated with ammonium sulfate (30% ographed on a Sephacryl S-500 (Pharmacia) column (2.6 X 90 cm, 16 saturation). After 15 h a t 4 "C, the ammonium sulfate precipitatewas ml/h) at room temperature in a 50 mM Tris-HC1 buffer, pH 7.4, centrifuged (15,000 X g, 30 min). The pellet was resuspended in 50 containing 0.5 M NaCl. The effluent was monitored at 214 nm, and mM Tris-HC1 buffer, pH 7.6, containing 0.2 M NaCl, 0.5% Nonidet 8-ml fractions were collected and stored at -20 "C. P-40, and 1mM EDTA. After stirring at 4 "C for 15 h,the suspension Cyanogen Bromide Cleauage-A crude 1 M NaCl tendon extract was centrifuged for 15 min in anEppendorf microcentrifuge, and the was precipitated with ammonium sulfate (40% saturation). After supernatant was dialyzed extensively against the same buffer at 4 "C centrifugation (12,000 X g, 20 min), thepellet was washed three times before it was used for immunoprecipitation. with 75% ethanolcontaining 1.0% potassium acetate and freezeThe cell pellet was resuspended in 50 mM Tris-HC1 buffer at pH dried. This material was resuspended in 70% formic acid and treated 7.6, containing 0.2 M NaC1,0.5% Nonidet P-40, and 1 mM EDTA with CNBr (12 mg/ml) under nitrogen for 4 h at 30 "C. The resulting (3.6 X lo7 cells/ml) and homogenized. The homogenate was stirred solution was diluted 10 times with water and freeze-dried. at 4 "C for 15 h and centrifuged (20,000 X g, 20 min). The proteins in Clostridiopeptidase A (EC 3.4.24.3) Digestion-The samples to be the supernatant were precipitated with ammonium sulfate (20-60% digested were either dialyzed against or dissolved in a 50 mM Tris- saturation). After 15 h at 4 "C, the ammonium sulfate precipitates HCl buffer, pH 7.6, containing 0.2 M NaCl and 5 mM CaC12. The were centrifuged (15,000 X g, 20 min). The pellet was resuspended in samples were then denatured by heating at 100 "C for 1min. Aliquots 50 mM Tris-HCI buffer, pH 7.6, containing 0.2 M NaCl, 0.5% Nonidet were cooled downand mixed with an equal volume of enzyme solution P-40, and 1 mM EDTA by stirring at 4 "Cfor 15 h. After centrifuga(Advance Biofactures) (250 units/ml) in the same buffer or buffer tion for 15 min in an Eppendorf microcentrifuge, the supernatant alone and made 10 mM in N-ethylmaleimide. After 5 h at 37 "C with was dialyzed against the same buffer and used for immunoprecipitaoccasional shaking, SDS was added to a final concentration of 2%. tion. The samples were boiled for 3 min and dialyzed against electrophoImmunoprecipitation-For immunoprecipitation of labeled type resis sample buffer. XI1 collagen, we used a procedure involving preformed immune Chondroitin ABC Lyase (EC 4.2.2.4) Digestion-The sample was complexes, as described by Tolleshaug et al. (16). The complexes were suspended in a 0.25 M Tris-HC1 buffer, pH 7.3, containing 0.25 M formed by incubating ascites fluid containing the monoclonal antisodium acetate. Chondroitinase ABC (Sigma) (20 milliunits in the body 75d7 with goat anti-mouse IgG (Organon Teknika) at 4 'C for same buffer) was added. The sample was incubated at 40 "C for 20 h 2 days. After centrifugation and washing, the complexes were resusand thendialyzed against the electrophoresis sample buffer. pended in 50 mM Tris-HC1 buffer, pH 8.0, containing 0.2 M NaCl, Gel Electrophoresis-SDS-polyacrylamide slab gel electrophoresis 0.5% Nonidet P-40, 1 mM EDTA, and kept at 4 "C. was performed in 7-cm X 8-cm X 0.75-mmgels ina mini PROIn preliminary experiments, 100-pl aliquots of preformed immune TEAN I1 (Bio-Rad) according to Laemmli (14) using 6 or 15% acryl- complexes were incubated with aliquots of media from fibroblast amide concentration. For reduction, samples were treated with 1%S- suspension cultures and labeled with [3H]proline.After centrifugation mercaptoethanol. The gels were stained with 0.1% (w/v) Coomassw through a sucrose gradient as described (16), the pellet was washed Brilliant Blue R-250 (Bio-Rad) in methanol/acetic acid/water and resuspended in electrophoresis sample buffer containing 0.5% p(40:1050) and destained with methanol/acetic acid/water (40:1050). mercaptoethanol. After boiling for 5 min, total radioactivity was determined by liquid scintillation counting. For gel electrophoretic analysis of immunoprecipitated proteins, Sugrue, S. P., Gordon, M. K., Seyer, J., Dublet, B., van der Rest, aliquots of immune complexes were incubated with aliquots of amM., and Olsen, B. R. (1989) J. Cell Biol., in press. monium sulfate-precipitated proteins from culture media or cell exThe abbreviations used are: SDS, sodium dodecyl sulfate; PVDF, tracts (see above). After centrifugation through sucrose, the pellets polyvinylidene difluoride; ConA, concanavalin A; CAPS, 3-(cyclo- were dissolved and examined by SDS-polyacrylamide gel electrophohexy1amino)-1-propanesulfonicacid. resis in 5% gels.

will be reported elsewhere.' In this paper, we describe the structure of the intact form of type XI1 collagen, based on immunoblots and affinity purification using the antibody. We also present rotary shadowing images of purified molecules that reveal a new structure among the constituents of the extracellular matrix.

Structure of Type XII Collagen

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Affinity Chromatography-Ascites fluid was centrifuged for 60 min a t 200,000 X g in a SW41 rotor (Beckman). The supernatant was dialyzed against the coupling buffer (0.1 M NaHC03, pH8.3, containing 0.5 M NaCI). CNBr-activated Sepharose 4B (Pharmacia) (0.4 g) was swollen for 15 min in 1 mM HCl and washed as suggested by the supplier. The dialyzed ascites fluid (10 ml, 0.42 mg/ml) was mixed with 1.4 ml of CNBr-activated Sepharose 4B and incubated in an end-over-end mixer during 2 h a t room temperature. The remaining active groups were blocked by a 2-h incubation with 0.1 M Tris-HC1, pH 8.0. The gel was then poured in a column, washed with 10 volumes of phosphate buffer (10 mM NaH2P04, pH 6.8, 0.15 M NaCI), and stored a t 4 "C in the presence of 0.04% of NaN3. Before use, the gel was equilibrated with 10 volumes of phosphate buffer. The CNBr peptides of the crude collagen preparation (6 mg/ml), dissolved in the same buffer, were applied to the gel a t a flow rate of 5 ml. cm-*. h". The gel was washed with 5 volumes of phosphate buffer. The flow was reversed, andthe gel was washed again with 5 volumes of phosphate buffer. The retained material was then eluted with 0.15 M sodium acetate at pH 3.5. The eluted fraction was dialyzed against 0.5 M acetic acid, concentrated, and analyzed by SDS-polyacrylamide gel electrophoresis. Sequencing of Blotted Peptides-The affinity-purified CNBr peptides were separated by electrophoresis on a 15% polyacrylamide gel and blotted onto anImmobilon" membrane as described above. After staining of the transferred peptides with Coomassie Blue, the bands were cut with a scalpel blade and submitted to repetitive Edman degradation as described by Matsudaira (17) in an AB1 model 470A instrument. The phenylthiohydantoin derivatives of the released amino acids were analyzed on-line with an AB1 model 120A PTHanalyzer. Rotary Shadowing-Proteins were dialyzed against 0.2 M NH4HC03a t a concentration of 30 pg/ml and mixed with an equal volume of glycerol immediately before spraying onto freshly cleaved mica discs. Shadowing was performed as described (18)on a rotating table with platinum a t a 9" angle, followed by coating with carbon a t a 90" angle. Electron micrographs were taken a t 80,OOOX magnification in a Philips EM 300 electron microscope.

RESULTS

Fractionation of Tendon Extracts-As described elsewhere,' immunoblots of 1M NaCl extracts of 17-day-oldchick embryo

leg tendons using the monoclona! antibody 75d7 demonstrated that the antibody recognizes a major band at M , = 220,000, using globular molecular weightmarkers, and a second band of higher molecular weight(data notshown). Without reduction, the immunoreactivity was only detected in the stacking gel. The immunoreactive bands disappeared after collagenase digestion. Since the immunoblot of the tendon extract revealed thatthe molecularweight of theintact al(XI1) chain is much larger than expected for a homologue of type IX collagen, we systematically monitored the purification of type XI1 collagen bydot immunoblots. The most effective extraction procedure was 1M NaCl in a 50 mM Tris-HC1 buffer. After precipitation of the extracted material with ammonium sulfate at 40% saturation, it was impossible to resolubilize the immunoreactive type XI1 molecule with 0.5 M HACor l M NaCl in Tris-HC1 buffer. In the presence of 0.2% SDS and after heat denaturation, the precipitate could be solubilized. A portion of this material, corresponding to the extract of the tendons of 240 embryos, was applied to a Sephacryl S-500 column and chromatographed in the presence of 0.2% SDS. The column was monitored by dot immunoblots of the fractions, using anti-type VI and antitype XI1 collagenantibodies, as theproperties of intact type XI1 collagen appeared similar to those of type VI ( M , of the reduced chains, presence of disulfide bonds). The Sephacryl S-500 column resolved almost completely type VI and type XI1 collagens (Fig. l),as shown by the dot blots reproduced at thecenter of the figure. Weconsistently observed a weaker reaction with the type XI1 antibody compared to thetype VI antibody. The fractions containing type XI1 collagen (41-51) showed two major bands, co-migrating with the bands detected with the 75d7 antibody. A fainter additional band comigrated with the al(1)collagen chain and was indeed shown to correspond to this chain by in situ CNBr cleavage and , I .. . I :i ,!

I !

11

1:

I I

~lJLJ"'i"'.":!-

t

. . _

"

cmml -11sml CWAta*

L

-m)lta*

Frictions:

FIG.1. Gel electrophoresis of the fractions of the Sephacryl S-500 column done under denaturing conditions. T w o 300-pl aliquots of each fraction have been analyzed on 6% polyacrylamide gels. Top, unreduced; bottom, reduced with 1%8-mercaptoethanol. A t the center of the figure, two rows of dot immunoblots show the reactivity of each fraction with a monoclonal antibody against type VI collagen (kindly given by Dr. Richard Mayne, University of Alabama in Birmingham) (upper row) and with the 75d7 antibody (lower row). Molecular weight markers are shown at the extremities of the gels;left, pepsin-treated type I collagen; right, globular standards. Bands inthe gel were visualized with Coomassie Blue.

Structure of Type X I I Collagen electrophoresis in a second dimension (data not shown). An immunoblot of these fractions confirmed the identity of the type XI1 collagen bands (Fig. 2). Alternatively, type XI1 collagen could be partially purified and concentrated without denaturation by affinity chromatography on a ConA-Sepharosecolumn. The 1M NaCl extract from the tendons of 100 embryos was applied to the column as described under “Materials and Methods.” Type XI1 collagen bound to thematrix and was eluted with 1 M methyl aD-glucopyranoside. The fractions containing type XI1 collagen (total volume, 20 ml) were applied as such to a Sephacryl S-500 column run without detergent. Fractions highly enriched in type XI1 collagen were obtained (Fig. 3). On this column, type XI1 spread over a larger number of fractions than when chromatography was carried out under the denaturing conditions shown in Fig. 1. The reasons for this are not clear. Type XII Collagen Is Not a Proteoglycan-Since type IX collagen carries a glycosaminoglycan side chain attached to its NC3 domain (19, 20), we wanted to determine whether this is also the case for type XI1 collagen. Chondroitinase ABC digestion of a crude extract or of a fraction purified by Sephacryl S-500 chromatography did not modify the electrophoretic mobilities of immunostained or Coomassie Bluestained type XI1 collagen bands. Furthermore, Alcian blue failed to stain the fractions enriched in type XI1 collagen under conditions which strongly stain PGII, a proteoglycan which contains at most two glycosaminoglycan side chains (21) per core protein. Type XZZ Collagen Is a Homotrimer-The cDNA-derived

sld443733951

FRACTION NUMBERS 51

56

Non-reduced ” ” ” ” -

-

4200k[k

a m -m

-

C 116

C 97.4

k[k kCh

662 kCh

Reduced

C 116 C 97.4 kCh

4 662

Fractions: std

kCh

36 37 30 39 40 41 42 43 std

FIG. 3. Gel electrophoresis of fractions obtained from the nondenaturing SephacrylS-500 chromatography. T w o 500-pl aliquots of the fractions indicated at the bottom of the figure have been analyzed on 6% polyacrylamide gels. Top, unreduced; bottom, reduced with 1% 6-mercaptoethanol. Molecular weight markers are shown at theertremities of the gels; left, pepsin-treated type I collagen; right, globular standards. Bands in the gel were visualized with Coomassie Blue.

std

al(I)4

a2(1)4

13153

0-118km C WA-

al(l)a a2(I) -e

6 e a 2 L D I

sequence of al(XI1) predicts the presence of methionyl residues at positions 15, 24,40, and 139, counted from the carboxyl end of al(XI1) (ll).’As shown earlier, CNBr cleavage of the molecule yieldeda fragmentof 11kDa (99 residues) perchain comprising the epitope recognizedby the 75d7 monoclonal antibody and 2 cysteinyl residues which must be involved in interchain disulfide bridges. A partial cleavage product of 14 kDa was also observed. Unreduced, multiple bands around M , = 30,000 were observed, probably due to various combinations of partial cleavage products. An affinity column was made with the 75d7 antibody. An unreduced CNBr digest of a crude 1 M NaCl extract from chick embryo tendons was then passed through the column. The retained material showed by electrophoresis the same multiple bands around M , = 30,000 that were observed by immunoblotting of the CNBr digest (Fig. 4). Protein bands in a similar gel were transferred onto a PVDF membrane. The transfer was stained with Coomassie Blue and the most intense band (second from the bottom) was excised and submitted to sequential Edman degradation. A single sequence was obtained, identical with that predicted by the cDNA pMG377 (ll),except for hydroxylation of some lysyl and prolyl residues:

Hyp-Gly-Glu-Hyl-Gly-Glu-Arg-Gly-Thr-Gly-SerGln-Gly-Pro-Arg-Gly-Leu-Hyp-Gly-Pro-

FIG. 2. Immunoblot ofselected fractionsfrom the Sephacryl 5-500 column done under denaturing conditions.Two 100-p1 aliquots from the fractions indicated on top of the figure were run on 6% polyacrylamide gels after reduction with 1%8-mercaptoethanol and stained with Coomassie Blue (upper panel) or immunostained with the 75d7 antibody (lower panel). Molecular weight markers are shown at theextremities of the gels; Left, pepsin-treated type I collagen; right, globular standards.

The remaining bands were also excised, pooled, and sequenced. The resulting sequence was identical to the one shown above. Only one sequence was thus obtained from the unreduced disulfide-bonded fragment. Biosynthetic Studies-To determine whether type XI1 collagen undergoes extensive modification during biosynthesis

Structure of Type XII Collagen

13154

1

2

3

4

1 2 3 38.5 kDa 25 kDa 24 kDa 6 175kDa 4- 13.5 kDa

4

5

6

7

4-

*

Case

9

200 kDa W



+ -

FIG. 4. Gel electrophoresisof the CNBr peptideof type XI1 collagen containing the epitope of the 75d7 antibody. Lanes 1 and 2 were loaded with 40 pg of a CNBr digest of a 1 M NaCl tendon extract, blotted onto an Immobilon” membrane, and immunostained with the antibody 75d7. The sample on lane 2 was digested with collagenase (Case). Lane 3 was loaded with the unreduced affinitypurified CNBr peptide and stained with Coomassie Blue. Lane 4 shows type I collagen CNBr peptides used as molecular weight standards.

or secretion, chick embryo tendon fibroblasts were incubated in the presence of [3H]proline,and radiolabeled proteins were immunoprecipitated with the monoclonal antibody 75d7. As shown in Fig. 5, the antibody precipitates proteins migrating at about M,= 200,000, as well as a band of higher molecular weight, both from culture media and in cell extracts. These radioactive bands were not observedwhenmedia samples were treated with bacterial collagenase before incubation with the preformed immune complexes (data not shown). The Globular Domain of Type XZZ Collagen-An aliquot of the Sephacryl S-500 column fractions containing type XI1 collagen wasdigested with bacterial collagenase, as described under “Materials and Methods.” After reduction, a single band, with a molecular mass of 190 kDa, based on globular standards, was seen by gel electrophoresis (Fig. 6). Without reduction, the peptide remained in the stacking gel. A band of the same molecular mass was seen when immunoprecipitated, radiolabeled material was digested with bacterial collagenase and analyzed by gel electrophoresis (data notshown). Visualization by Rotary Shadowing-Fraction 43 from the Sephacryl S-500 column run under denaturing conditions (Fig. 1) was dialyzed overnight against 0.2 M (NHJHC03, allowing partial renaturation of the molecule, and analyzed by rotary shadowing. A large proportion of the visualized molecules had a “lollipop” appearance with a short, approximately 75 nm long tail and alarge globular domain (Fig. 7A). The thin tail often appeared kinked. After collagenase digestion, only globules were observed(not shown). When fraction 40 from the nondenaturing Sephacryl S-500 column (Fig. 3) was analyzed, the molecules appeared as crosses with one thin 75 nm tail anda central globular domain from which three 60 nm thicker arms extended (Fig. 7B). A smaller globule was also apparent at theextremity of each arm. After heat denaturation and renaturation of this fraction, molecules with a large globule and a thin tail were observed, similar to those shown in Fig. 7A. They often formed large aggregates. DISCUSSION

The identification of type XI1 collagen in this work is based on the specificity of the monoclonal antibody 75d7 which was

FIG. 5. Gel electrophoresis of radiolabeled and immunoprecipitated type XI1 collagen. Tendon fibroblasts were incubated in suspension culture in thepresence of [3H]proline,and labeled proteins were analyzed after immunoprecipitation with the monoclonal antibody 75d7 as described under “Materials and Methods.” All samples were reduced before electrophoresis in a 5% gel. The positions of globular molecular weight markers are indicatedon the left. For fluorography, lane 1 was exposed for 1 week, while lanes 2-7 were exposed for 3 weeks. Lane 1, aliquot of ammonium sulfate-precipitated medium proteins (13,000cpm). Lune 2, same asin lane 1, except that the material was treated with 1 mg/ml pepsin in 0.5 M acetic acid a t 4 “C overnight, before being loaded on thegel. Lanes 3 and 4, aliquot of ammonium sulfate-precipitated medium proteins (1.6 X 10’ cpm), immunoprecipitated with preformed immune complex. In an attempt to release the radiolabeled material from the immune complex without dissolving the complex itself, the precipitate was treated first with 0.02 M acetic acid for 20 s. The acetic acid-extracted material was loaded in lane 4, while the remaining pellet was dissolved in sample buffer and loaded in lane 3. Lanes 5 and 6,aliquot of an ammonium sulfate-precipitated cell extract (10‘cpm) immunoprecipitated with preformed immune complex. The precipitate was treated with 0.02 M acetic acid for 20 s. The acetic acid-extracted material was loaded in lane 6,while the material in the remaining pellet was run in lane 5.

raised against a synthetic peptide made on the basis of the sequence of the cDNA pMG377. The sequence reported here of an immunopurified CNBr peptide further supports the specificity of this antibody. The studied Sepharose S-500 fractions contained type XI1 collagen, as shown by immunoblots (Fig. 2), and the electrophoretic patterns of these fractions show the presence of molecules with molecular weights and collagenase sensitivity identical to immunopurified type XI1 collagen. However, from our data, we cannot conclude with certainty that XI1 collagen is the sole collagen component in the column fractions. In fact, we have preliminary evidence that, at least in mammals, a molecule very similar to but genetically distinct from type XI1 collagen is present in some type I-containing mat rice^.^ It is therefore possible that more than one genetic type of molecule is present in these fractions. Type XI1 collagen was initially discovered during the search for a type IX collagen homologuein type I-containing tissues (11).The data presented here demonstrate that these two collagen types are actually very different insize and thattype XI1 collagen presents an as yet undescribed macromolecular structure. The rotary shadowing pictures (Fig. 7) and the B. Dublet and M. van der Rest, unpublished observation.

Structure of Type XII Collagen

std

1

std

2

200kDa +

-6

i

116 kDa --t

i

97.4 k h -

662 kDa

-

Case

P -Ski

190 kDa

al(1) a2(I)

- + + +

FIG.6. Collagenase digestion of type XII collagen. A 1-ml aliquot from fraction 43 of the Sephacryl S-500 chromatography (Fig. 1) was digested with clostridiopeptidase A and analyzed by electrophoresis as described under “Materials and Methods.” The samples were reduced with 8-mercaptoethanol prior to the electrophoresis. Lune I , undigested control; lane 2, digested sample. Molecular weight markers are shown at theextremities of the gel; right, pepsin-treated type I collagen; left, globular standards. Bands in the gel were visualized with Coomassie Blue.

A

B

FIG. 7. Rotary shadowing electron micrographsof type XII collagen. A, gallery of selected molecules observed in fraction 43 from the Sephacryl S-500 chromatography done under denaturing conditions (Fig. 1).The molecules were allowed to partially renature during an overnight dialysis a t 4 “C against ammonium bicarbonate. B, gallery of undenatured molecules observed in fraction 40 from the chromatography shown on Fig. 3. Bar = 100 nm.

collagenase digestion of intact type XI1 (Fig. 6) suggest that the triple helical region is smaller than in type IX while the non-triple helical amino-terminal domain is much larger. The sequence analysis of extended cDNAs, that will be discussed in a parallel paper,’ indeed reveals that al(XI1) collagen contains only two triple helical domains which correspond to the 16- and 10-kDa reduced pepsin-resistant fragments described earlier (12). The size of the collagenase-sensitive tail

‘M. K. Gordon, D. R. Gerecke, B. Dublet, M. van der Rest, and B. R. Olsen, manuscript in preparation.

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and the location of the kink as determined from the rotary shadowing images are in perfect agreement with the DNA data. The sequence analysis of the unreduced, immunopurified CNBr peptide suggests that type XI1 collagen is a homotrimer. The rotary shadowing images showing three thick arms of identical length and appearance, as well as previously reported data on bovine type XI1 pepsin-resistant fragments further support this conclusion. This differentiates type XI1 from type IX collagen, which is a heterotrimerof three genetically distinct chains (22). One of the most remarkable features of type IX collagen is the presence of a glycosaminoglycan side chain attached to a specific sequence in the NC3 domain of the a2(IX)chain (19, 20). Type XI1 collagen is not a proteoglycan since its electrophoretic mobility is not affected by chondroitinase ABC digestion and since it does not stainwith Alcian blue. However, its binding to Con A-Sepharose indicates that itis a glycoprotein. The natureof its carbohydrate moiety has not yet been investigated. Type XI1 collagen always appeared as two major bands on electrophoretic gels, one at M , = 220,000 and the second appearing as a doublet at M, = 270,000 and 290,000. These multiple bands have been observed byimmunoblots and Coomassie Blue staining. Bands of identical mobilities have also been seen in immunoprecipitated radiolabeled material, indicating that type XI1 collagen undergoes little, if any, proteolytical processing in tissues. After bacterial collagenase digestion, a major band a t 190 kDa is seen. We have done several experiments to try to unravel the natureof the higher molecular weight bands; culture of cells in the presence of 8aminopropionitrile to prevent lysine-derived cross-linking, treatment with periodic acid to break Schiff bases, digestion with N-glycanase to eliminate N-linked oligosaccharides and two-dimensional electrophoresis with in situ CNBr cleavage before the second dimension to detect possible aggregates with fibrillar collagens. None of these approaches was conclusive. While type I collagen CNBr peptides were clearly identifiable in these two-dimensional gels, we could not rule out that they could have arisen from contaminating type I 0 and y components. In addition, intact type XI1 collagen does not produce a clearly defined pattern of bands after CNBrcleavage, probably due to its size and thepresence of carbohydrate. This furthercomplicates the interpretation of such data. The pepsin fragmentationpattern (12) andthe cDNA sequenced indicate that the triple helical domains, the nontriple helical sequence between them,andthe non-triple helical sequence at thecarboxyl end accountfor about 35 kDa of the molecular mass of al(XI1). This is in excellent agreement with the appearance of a band at 190 kDa after treatment with bacterial collagenase. Since fractions containing both 220-kDa and 270-290-kDa bands of type XI1 collagen give rise to only one high molecular weight band at 190 kDa after collagenase treatment, we hypothesize that the additional molecular weight of the latter form is due to thecrosslinking of collagenase-sensitive material, for example, fibrillar collagen chains, to type XI1 collagen. Since the molecular weights are based on relative mobilities on polyacrylamide gels and since collagenous peptides are known to migrate differently from globular proteins, the values estimated for M , are only tentative. Additional work willclearly be required to fully explain this highly reproducible electrophoretic pattern. The structureof the non-triple helical amino-terminal domain revealed by rotary shadowing is reminiscent of that of several adhesion glycoproteins such as fibronectin, laminin, and brachionectin/tenascinthat all show the presence of non-

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Structure of Tyi,e XII Collagen

triple helical arms separating smallglobules (23,24). Whether this structural similarity is a reflection of primary structure and function homology can only be answered by further studies. It is interesting to note that molecules with shapes similar to thatof our type XI1 collagen preparation have been observed in a crude tendon extract.' This indicates that the shape of the molecules as reported here have not been altered by the preparation procedure. The data presentedheredonot allow firm conclusions about the function of type XI1 collagen. However, we speculate that type XI1 molecules are associated with type I collagen-containing fibrils. This is based on co-localization of the two collagens in many tissues' and on the sequence similarity between the carboxyl-terminal COLI domain of al(XI1) and the corresponding domain in type IX collagen. This similarity is particularly striking when one compares the al(XI1) and al(1X) collagen chains. For example, the COLl domains of both chains contain two imperfections in the Gly-Xaa-Yaa tripletstructure in the same relative locations, and both chains contain 2 cysteinyl residues at the end of the triple helix. In addition, the overall amino acid sequence similarity of the COLl domains between the two chains is almost as high as the similarity between the al(1X) and a2(IX) chains in the same region. Finally, analysis of the exons encoding the COLldomain in the al(XI1)gene shows that this partof the gene is homologous to the corresponding parts of the al(1X) and aZ(1X) collagen genes. This suggests that the COLl domain plays asimilar role intypes IXand XI1 collagens, and we believe that role is to participate in binding to type 11- and type I-containing fibrils. AcknowZedgments-We want tothank Dr. Richard Mayne for providing the anti-type VI collagen antibody, for his help in establishing the rotary shadowing technique and for stimulating discussions, Dr. Graham Greene for suggesting the use of ConA-Sepharose for purifying type XI1 collagen, H. Lecavalier and G. Charette for technical help, and M. Lepik for the illustrations.

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