Matrilin-2, a Large, Oligomeric Matrix Protein, Is Expressed by aGreat Variety of Cells and Forms Fibrillar Networks*

(Received for publication, November 12, 1998, and in revised form, January 27, 1999)

Dorothea Piecha‡, Selen Muratoglu§, Matthias Morgelin¶, Nik Hauser‡, Daniel Studeri,Ibolya Kiss§, Mats Paulsson‡, and Ferenc Deak§**

From the ‡Institute for Biochemistry, Medical Faculty, University of Cologne, D-50931 Cologne, Germany, the §Institute ofBiochemistry, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary, the¶Department of Cell and Molecular Biology, Lund University, S-22100 Lund, Sweden, and the iM. E. Muller-Institute forBiomechanics, CH-3010 Bern, Switzerland

Matrilin-2 is a member of the protein superfamily withvon Willebrand factor type A-like modules. Mouse ma-trilin-2 cDNA fragments were expressed in 293-EBNAcells, and the protein was purified, characterized, andused to immunize rabbits. The affinity-purified anti-serum detects matrilin-2 in dense and loose connectivetissue structures, subepithelial connective tissue of theskin and digestive tract, specialized cartilages, andblood vessel walls. In situ hybridization of 35S-labeledriboprobes localizes the matrilin-2 mRNA to fibroblastsof dermis, tendon, ligaments, perichondrium, and peri-osteum; connective tissue elements in the heart; smoothmuscle cells; and epithelia and loose connective tissuecells of the alimentary canal and respiratory tract. RNAblot hybridization and immunoblotting revealed bothmatrilin-2 mRNA and protein in cultures of a variety ofcell types, confirming the tissue distribution. Alterna-tive splicing affects a module unique for matrilin-2 in allof the above RNA sources. SDS-polyacrylamide gel elec-trophoresis and electron microscopy reveals matrilin-2from tissue extracts and cell line cultures as a mixture ofmono-, di-, tri-, and tetramers. Matrilin-2 is substitutedwith N-linked oligosaccharides but not with glycosami-noglycans. Because of other, yet unidentified, cell-typedependent posttranslational modifications, the mono-mer is heterogeneous in size. Immunofluorescenceshowed that matrilin-2 functions by forming an extra-cellular, filamentous network.

Extracellular matrix provides physical support to the cells,delineates pathways for cell migration during differentiationand tissue regeneration, and provides the necessary milieu forthe normal cell metabolism and development. Collagen fibersand proteoglycan aggregates provide the structural basis formatrix architecture. Noncollagenous proteins modulate the or-ganization of these elements, form collagen-associated or inde-pendent networks, and are parts of cell migratory pathways.

The matrix molecules share homologous modules, proteindomains of common evolutionary origin, but a great functionalvariability of the homologous modules in different proteins hasbeen observed. The recently discovered matrilins (for a review,see Ref. 1) are typical modular proteins belonging to the super-family with von Willebrand factor type A-like (vWFA)1 mod-ules. Members of the matrilin family are found in a widevariety of extracellular matrices. Matrilin-1, formerly calledcartilage matrix protein, and matrilin-3 (2, 3) are abundant incartilage, while matrilin-2 (4) and matrilin-4 (5) show a broadertissue distribution. Thus, all forms of connective tissue appearto contain at least one form of matrilin, indicating a generaland important function for this protein family.

Matrilin-2 was found to contain the same protein modules inthe same order as matrilin-1 (4). The precursor protein inmouse is 956 amino acids long and consists of a putative signalpeptide, two vWFA domains connected by 10 epidermal growthfactor-like modules, a potential oligomerization domain, and aunique segment. The ability of the 38 C-terminal amino acidmoieties to form an a-helical coiled-coil was shown by Pan andBeck (6). Matrilin-2 mRNA was detected by filter hybridizationin a variety of mouse organs including calvaria, uterus, heart,and brain as well as fibroblast and osteoblast cell lines. A groupof 120–150-kDa bands was, after reduction, recognized specif-ically with an antiserum against the matrilin-2-glutathioneS-transferase fusion protein in media of the matrilin-2-express-ing cell lines. Immunolocalization of matrilin-2 in developingskeletal elements showed reactivity in the perichondrium andthe osteoblast layer of trabecular bone.

In order to gain a better understanding of the potentialfunction of matrilin-2, we have determined the spatial expres-sion of the gene by radioactive in situ hybridization. A newantiserum with a higher titer to the native matrilin-2 wasraised using, as an antigen, matrilin-2 expressed in a eukary-otic cell line, and the protein was immunolocalized in mousetissues. Furthermore, matrilin-2 was purified from media ofcells overexpressing the full-length protein and visualized byelectron microscopy to provide information on the moleculardimensions and oligomeric structure of the protein. The struc-tural information was extended by SDS-PAGE analysis of theintact protein and the reduced subunits. Posttranslationalmodification of the protein and alternative splicing of themRNA were also characterized. Finally, formation of an extra-cellular network in cultures of cells expressing matrilin-2 wasdemonstrated by indirect immunofluorescence. The potential

* This work was supported by joint grants from the Volkswagen-Stiftung (I/71 654) and the Bilateral German-Hungarian CooperationProgram (D-10/96/WTZ), Hungarian National Scientific ResearchFoundation Grants OTKA T023803 and T029157, Deutsche Forsch-ungsgemeinschaft Grant Kr 558/10–3, and a grant from the Koln For-tune program of the Medical Faculty of the University of Cologne. Thecosts of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

** To whom correspondence should be addressed: Institute of Bio-chemistry, Biological Research Center, Hungarian Academy of Sci-ences, P.O. Box 521, H-6701 Szeged, Hungary. Tel.: 36-62-432-232; Fax:36-62-433-506; E-mail: matrix@nucleus.szbk.u-szeged.hu.

1 The abbreviations used are: vWFA, von Willebrand factor typeA-like; EGF, epidermal growth factor; PAGE, polyacrylamide gelelectrophoresis.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 19, Issue of May 7, pp. 13353–13361, 1999© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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function and suggested molecular architecture of the protein isdiscussed.

MATERIALS AND METHODS

Mouse Strains and Cell Cultures

BALB/c or NMRI mouse strains were used for RNA or protein anal-ysis, respectively. The mouse fibroblastic cell lines WEHI 164 and NIH3T3, the rat osteogenic sarcoma UMR-106, and the small intestineepithelial IEC-6 cell lines were obtained from the American Type Cul-ture Collection (Rockville, MD). The mouse C2/7 cells with skeletalmuscle characteristics (7), the smooth muscle-like cell 9E11G (8), thekeratinocyte carcinoma PVD(A)I (9), the rat Schwann cell line RN22(10), and the Swarm rat chondrosarcoma cell line (RCSC) (11) wereobtained from the laboratories of origin. The mouse immortalized en-dothelial cells m1END, derived from mesenteric lymph nodes (12), andthe SV40-transformed lymphoid vascular endothelial cell SVEC (13)were provided by L. Sorokin and R. Hallmann (Erlangen); smoothmuscle cells (SMC) from rat aorta were cultured by F. Michaelsen(Cologne), using standard methods (14). Unless recommended other-wise by the supplier, the cell lines were cultivated in Dulbecco’s modi-fied Eagle’s medium supplemented with 10% fetal calf serum (LifeTechnologies, Inc.), and utilized for RNA and protein analyses.

RNA Preparation and Analysis

Total RNA was prepared from guanidinium thiocyanate extracts ofvarious cell lines using the RNA isolation kit of Stratagene. For RNAblot analysis, 7-mg aliquots were electrophoresed, blotted to Hybond Nfilter (Amersham Pharmacia Biotech) and hybridized consecutivelywith pCRP12 cDNA (4) and chicken 27 S rRNA gene fragment. For thestudy of RNA alternative splicing, total RNA was reverse transcribedwith Moloney murine leukemia virus reverse transcriptase (Life Tech-nologies) using oligo(dT) primer. Nested polymerase chain reactionswere performed using first the distal primers 59-GGACGGGCTCAG-GATGA-39 and 59-CTGTATCTCAGGCGATTTTC-39 and then the prox-imal primer pair 59-CATTGACAAGCATCTCTTCT-39 and 59-TTTG-TGTAGACCGTGAAAGA-39 flanking the unique region of mousematrilin-2.

In Situ Hybridization

Preparation of Tissue Sections—For paraffin embedding, tissue spec-imens were fixed overnight in 95% ethanol, 1% acetic acid; dehydratedin ethanol; cleared in xylol; and embedded in low melting point paraffin(Paraplast, Sigma). Sections of 7–9 mm were cut and mounted onpoly-L-lysine-coated slides. For cryostat sections, specimens were fixedovernight in 4% paraformaldehyde, 8 mM NaHPO4, 0.15 M NaCl, pH 7.4;decalcified, if necessary, in 15% EDTA, 2% paraformaldehyde, 4 mM

NaHPO4, pH 7.4; and embedded in Tissue-Tek O.C.T. compound(Sakura Finetek Europe). 12-mm sections were cut.

Hybridization—For pretreatment, in situ hybridization, and wash-ing, the protocol as outlined by Hofstetter et al. (15) was used. Briefly,sections were deparaffinized and rehydrated, if necessary, and thendigested with proteinase K (1 mg/ml), postfixed, and acetylated in 0.25%acetic anhydride. Riboprobes were labeled with [35S]CTP and hydro-lyzed to 150-nucleotide average length. The sections were hybridized for12–16 h at 53 °C with riboprobes at a final activity of 1–4 3 107 cpm/ml,depending on their length. After hybridization, the tissue sections werewashed at 53 °C in 50% formamide, 2 3 SSC, 1 mM EDTA, 10 mM

dithiothreitol; treated with RNase T1 (1 unit/ml); and washed again at53 °C in 50% formamide, 0.23 SSC, 1 mM EDTA. The slides weredehydrated and dipped in LM1 photoemulsion (Amersham PharmaciaBiotech). Autoradiography was performed for 5–10 days, and sectionswere counterstained in Mayer’s hematoxylin (Merck).

Expression and Purification of Recombinant Matrilin-2

Partially overlapping cDNA fragments in the mouse matrilin-2clones pCRP207, pCRP190, and pCRP12 (4) were combined to full-length cDNA using suitable restriction enzymes. One AflII site wasinserted immediately upstream of the first AUG codon by polymerasechain reaction. After digestion with AflII and NotI, a 3.3-kilobase paircDNA fragment was inserted into the expression vector pCEP-Pu (16),cleaved previously with AflII and NotI. The resulting clone, pCEP-Mtr2, encoded the full-length matrilin-2 precursor, including the secre-tion signal peptide.

The recombinant plasmid was introduced into the human embryonickidney cell line 293-EBNA (Invitrogen), which constitutively expressesthe EBNA-1 gene product from Epstein-Barr virus. The transfectedcells were selected with 1 mg/ml puromycin and grown to confluency.

Secretion of matrilin-2 into the culture medium was verified by SDS-PAGE and immunoblotting, using antiserum against a matrilin-2-glu-tathione S-transferase fusion peptide (4). Serum-free culture mediumwas dialyzed against 2 M urea, 50 mM Tris-HCl, pH 8.6, and applied toa DEAE-Sepharose-FF column. The bound proteins were eluted with alinear gradient of 0.05–0.4 M NaCl. Fractions eluted between 0.1 and0.2 M NaCl were pooled, and matrilin-2 was further purified by gelfiltration through a Sepharose CL-4B column equilibrated in 2 M urea,150 mM NaCl, 50 mM Tris-HCl, pH 7.4. The final purification wasachieved on a Heparin-Sepharose column equilibrated in the samebuffer. Matrilin-2 bound exclusively to heparin and was eluted at about0.3 M NaCl.

Another cDNA fragment encoding the 10 EGF-like modules and thevWFA2 module of mouse matrilin-2 was inserted into the NheI–NotIsites of the pCEP-Pu vector, downstream of the secretion signal se-quence of BM40 (16). The cells were transfected, and the selection andcollection of media followed as mentioned above. The serum-free me-dium was diluted 3-fold with 50 mM Tris-HCl, pH 7.4, and applied to aQ-Sepharose-FF column. The bound proteins were eluted with a lineargradient of 0.05–0.4 M NaCl. Fractions containing the matrilin-2 frag-ment were refractionated on a Mono-Q fast protein liquid chromatog-raphy column and were apparently free of contaminants. The purifiedmatrilin-2 fragment was used to immunize rabbits. The antiserum waspurified by affinity adsorption to the antigen.

Immunoblotting of Cell Culture Media and Mouse Organ Extracts

Cell cultures were grown to confluency in Dulbecco’s modified Eagle’smedium supplemented with 10% fetal calf serum. The cell layers werewashed and cultured for 48 h without serum, and the medium washarvested. Several mouse organs were homogenized using a Polytronhomogenizer and extracted with 0.25 M NaCl, 50 mM Tris-HCl, pH 7.4,containing as protease inhibitors 10 mM EDTA, 2 mM N-ethylmaleim-ide, 2 mM phenylmethylsulfonyl fluoride. After centrifugation, aliquotsof the supernatant were analyzed. For immunoblotting, samples weresubmitted to SDS-polyacrylamide gel electrophoresis according to theprotocol of Laemmli (17), using gradient gels of 4–15% polyacrylamide.Proteins were transferred electrophoretically to a nitrocellulose filterand developed with affinity-purified antiserum to matrilin-2, followedby peroxidase-conjugated swine anti-rabbit IgG (DAKO) and the ECLchemiluminescence procedure (Amersham Pharmacia Biotech) as sug-gested by the suppliers.

Immunohistochemistry and Immunofluorescence of Cell Layers

Immunohistochemistry was performed as described previously (18),using the affinity-purified anti-matrilin-2 antiserum together with aswine anti-rabbit IgG-peroxidase complex and 3-amino-9-ethylcarba-zole as substrate on unfixed cryosections from adult and newbornmouse. For immunofluorescence of cell cultures, cells were plated ontoplastic chamber slides, and after reaching confluency they were fixed in2% paraformaldehyde in phosphate-buffered saline for 10 min. In someexperiments, cells were permeabilized by treatment with 10% NonidetP-40 in phosphate-buffered saline for 10 min. Nonspecific antibodybinding was blocked by incubation with 1% (w/v) bovine serum albuminin phosphate-buffered saline for 1 h. The cells were treated with theaffinity-purified antibody to matrilin-2 for 1 h followed by CyTM3-conjugated affinity-pure goat anti-rabbit IgG (Jackson ImmunoRe-search Laboratories). Pictures were taken with a Zeiss Axiophot micro-scope equipped with a fluorescence source.

Analysis of Posttranslational Modifications

The potential substitution with sulfated glycosaminoglycans wasdetermined by metabolic labeling. 293-EBNA cells transfected withpCEP-Mtr2 were grown in serum- and sulfate-free minimal essentialmedium containing 50 mCi/ml [35S]sulfate (Amersham Pharmacia Bio-tech). After a 48-h labeling period, media were harvested and precipi-tated with trichloroacetic acid (final concentration 12%). The radiola-beled proteins were separated by SDS-PAGE, and radioactive bandswere visualized by fluorography after treatment with 1 M sodiumsalicylate.

Chondroitinase ABC and Heparitinase Digestions—Cell culture me-dia from 293-EBNA cells transfected with pCEP-Mtr2 were incubatedwith 0.7 milliunits/ml heparitinase I (Sigma; from Flavobacterium he-parinum) and 1.7 milliunits/ml chondroitinase ABC (Sigma) for 7 h at37 °C. Aliquots of the digested and control media were analyzed afterSDS-PAGE and immunoblotting with specific antiserum to matrilin-2.

To test for the presence of N-glycosidically linked oligosaccharides,293-EBNA cells transfected with pCEP-Mtr2 were grown in serum-free

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Dulbecco’s modified Eagle’s medium for 48 h in the presence of tunica-mycin (Sigma) at 0.5 mg/ml. Media from tunicamycin-treated and con-trol cells were analyzed after SDS-PAGE and immunoblotting withspecific antiserum to matrilin-2. Parallel blots were developed withantibodies to nidogen, which is endogenously produced by the 293-EBNA cells and served as a positive control of better known glycosyla-tion (not shown).

N-Glycosidase F Digestion—Purified matrilin-2 in incubation buffercontaining 50 mM Tris-HCl, pH 7.4, 50 mM sodium chloride, 0.5%Nonidet P-40, and 0.1% SDS was denatured by heating at 100 °C for 2min. After denaturation, the protein was incubated with 0.3 units ofN-glycosidase F (Roche Molecular Biochemicals) per mg of protein for20 h at 37 °C. The control sample was treated similarly without addingN-glycosidase F. The digested and control samples were analyzed by4–15% SDS-PAGE and stained with silver nitrate.

Electron Microscopy

Purified matrilin-2 (10 mg/ml) was adsorbed to a 400-mesh carbon-coated copper grid, which was rendered hydrophilic by glow dischargeat low pressure in air. The grid was immediately blotted, washed withtwo drops of water, and stained with 0.75% uranyl formate for 15 s.Samples were observed in a Jeol 1200 EX transmission electron micro-scope operated at 60-kV accelerating voltage and 3 75,000 magnifica-tion. Images were recorded on Kodak ESTAR Thick Base 4489 plateswithout preirradiation at a dose of typically 2000 electrons/nm2. Eval-uation of the data from electron micrographs was done as describedpreviously (19).

RESULTS

Matrilin-2 Is Deposited in All Forms of Connective Tissue,Some Types of Smooth Muscle, and a Few Epithelia—A cDNAfragment encoding the EGF-like modules and the second vWFAdomain of mouse matrilin-2 was inserted into the pCEP-Puvector, utilizing the secretion signal sequence of BM40 (16).The recombinant plasmid was introduced into the 293-EBNAcell line, where it was stably maintained in episomal form. Thematrilin-2 fragment, secreted into the tissue culture medium,was purified and used to immunize rabbits. The antiserum,after affinity purification, specifically reacts with matrilin-2and does not show any cross-reactivity (see Fig. 5A).

Matrilin-2 was localized immunohistochemically in cryostatsections of adult and newborn mice (Fig. 1). The protein is mostabundant in dense connective tissue, including tendon, liga-ments, perichondrium, periosteum, dura mater, epineurium(Fig. 1, G–I); perimysium of skeletal, heart, and smooth muscle(Fig. 1, B, C, E, and G); submucosa of alimentary canal (Fig. 1,E and F); the reticular layer of dermis (Fig. 1, A and G); spleencapsule; and the annulus fibrosus, chordae tendineae, andvalves of heart (not shown). In loose connective tissue, localconcentration of the protein is not as high. It is most abundantin the papillary layer of dermis (Fig. 1A) and spleen trabeculae(not shown). It is less abundant, but detectable, in the laminapropria of alimentary canal and the tunica adventitia of bloodvessels and respiratory tract (not shown in detail). Matrilin-2 isalso detectable to variable extents in specialized connectivetissue, including the zones of proliferation and hypertrophy inepiphyseal cartilage (Fig. 1G), elastic cartilage of the ear (notshown), fibrocartilage in the annulus fibrosus of the interver-tebral disc (Fig. 1, H–I), and bone, where it lines the marrowcavities. The protein was abundant in the myometrium (notshown) and was also detectable between muscularis mucosaeand muscularis externa of the alimentary canal, possibly asso-ciated with the nervous plexus (Fig. 1E). The amount of theprotein was above the detectability threshold in a few special-ized epithelia, e.g. the sublingual gland in the newborn headand the lens epithelium or underlying basement membrane ofday 16.5 embryos (not shown). In nervous tissue, matrilin-2was observed in the dura and pia mater of brain and spinal cordas well as the perineurium of peripheral nerves (Fig. 1H).

The Matrilin-2 Gene Is Transcribed in Fibroblasts, Osteo-

blasts, Smooth Muscle Cells, and Some Epithelial Cells—Inorder to reveal where the matrilin-2 mRNA is produced, even-tually leading to extracellular deposition of the protein, weperformed in situ hybridization. Three antisense riboprobes,complementary to nonoverlapping regions of the matrilin-2mRNA were hybridized to cryostat and paraffin sections of5–10-week-old mouse. The hybridization of the radioactive ri-boprobe was detected by autoradiography and the silver grainswere visualized in dark field. Bright field photomicrographs ofthe same fields helped to identify the hybridizing tissues inthe sections (Fig. 2). Parallel sections were hybridized withsense riboprobes and verified the specificity of hybridization(not shown).

The results of in situ hybridization confirmed and extendedthe data obtained by immunohistochemistry. Connective tissuecells are clearly positive in dense and loose as well as special-ized connective tissue. Dense connective tissue fibroblastsshow characteristic accumulation of grains in tendon, liga-ments, perichondrium, periosteum (Fig. 2, A and B); cells in thereticular layer of dermis and at the base of hair papillae (Fig.2A); and annulus fibrosus of heart, atrioventricular valve, andchordae tendineae (Fig. 2D). Loose connective tissue cells alsogave hybridization signals in the adventitia of trachea (Fig. 2,E and G) and the mesentery cells (Fig. 2C). Matrilin-2 geneexpression was observed in epiphyseal cartilage, in the zones ofproliferation and early hypertrophy (Fig. 2B), as well as inosteoblasts of the calvaria (not shown).

Muscle cells showed a detectable level of gene expression,albeit not as high as that in fibroblasts. A strong in situ hy-bridization signal was observed in the organs where previousNorthern hybridization (4) showed a high steady state level ofmatrilin-2 mRNA. The uterus gave an overall strong hybrid-ization signal (Fig. 2C). Heart was also strongly positive, butwith a gradient toward the regions richer in connective tissuecells, e.g. the atria, auricle, valves, and chordae tendineae(Fig. 2D).

In addition to connective tissue cells and myoblasts, someepithelia also showed clearly positive hybridization signals. Inparaffin sections, the secretory epithelium of esophagus, themucosa, and serosa of colon as well as the seromucous glands oftrachea showed strong hybridization (Fig. 2, E–H).

Relative Abundance of Matrilin-2 mRNA in Established CellLines—In some cases it was difficult to determine with cer-tainty the cell types where the gene expression was observed byin situ hybridization. For example, smooth muscle cells are inclose association with fibroblasts, and epithelial cells form thinlayers in close proximity to the underlying connective tissue.Therefore, we examined matrilin-2 mRNA and protein produc-tion in homogeneous cultures of permanent cell lines.

Total RNA samples were isolated from cultured cells, and therelative amount of matrilin-2 mRNA was estimated by North-ern hybridization (Fig. 3). In all of the cell lines examined,expression of the gene was observed. We previously demon-strated that the fibroblastic cell lines L929, WEHI 164, NIH3T3, and the rat osteogenic sarcoma UMR-106 expressed thegene (4). In the present experiment, the mRNA level in NIH3T3 cells (Fig. 3, lane 1) was compared with other cell lines. Intwo samples, isolated from the rat chondrosarcoma cell line andthe 9E11G smooth muscle-like cells, the matrilin-2 mRNA levelwas higher than in NIH3T3 cells. The other smooth muscle cellline, SMC, isolated from rat aorta, and the differentiated skel-etal muscle myotube C2/7 contained less, but significant,amounts of matrilin-2 mRNA, confirming that the gene can beexpressed in cells with myoblast characteristics. The intestinalepithelial cell line IEC6 and the Schwannoma cell line RN22also expressed the gene at a detectable level. The least amount

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FIG. 1. Immunohistochemistry of mouse tissue. Sections from newborn (A, F, G, H, I, and J) and adult (B–E) animals were made, andmatrilin-2 (A–I) or matrilin-1 (J) was detected with affinity-purified antisera. A, in skin from the leg, strong immunostaining was observed in thepapillary layer (pl), and somewhat weaker staining was seen in the reticular layer (rl) of dermis and at the base of hair papillae (hp). ep, epidermis.B, in the heart, the auricle (au) is stained more intensely than the atrium wall (aw). C, in the ventricles, the connective tissue surrounding somecapillaries (ca) are positive, and staining of the basement membrane around myoblasts is weak. The interstitial connective tissue (ct) is stronglyreactive. D, in kidney, the arcuate arteries (aa) show strong staining. E, the esophagus epithelium (ep) is not stained, but the underlying basementmembrane and mucosa show strong immunoreaction. In the muscularis externa, connective tissue (ct) between the circular and longitudinalsmooth muscle cell layers shows strong immunostaining, possibly associated with the nervous plexus. F, in the oral cavity of a newborn mouse,fibers of periodontal membrane around the developing incisor (i) show strong staining as well as the submucosa of hard and soft palate and thelamina propria of the tongue (to). There is no staining in the epithelial layers (ep). G, in the ossifying skeleton of the leg, the perichondrium (pc),

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of matrilin-2 mRNA was found in the keratinocyte carcinomaPVD(A)I. In a separate experiment, matrilin-2 mRNA wasdetected in the SVEC endothelial cell line (not shown). Insummary, not only the connective tissue cell types containedmatrilin-2 mRNA, but gene expression was also observed inmyoblasts and in the epithelial and endothelial cell linestested.

Previous analysis indicated that the sequence variabilitywithin the unique region may be a consequence of alternativesplicing. Therefore, we performed reverse transcription-poly-merase chain reaction analysis to determine if there is furthermRNA heterogeneity within the translated region. In the

SVEC endothelial and the rat chondrosarcoma cell lines, alter-native splicing affected only the middle third of the uniquemodule but not the region encoding the coiled coil, the vWFA2,or EGF-like modules (data not shown). Systematic comparisonof the RNA samples showed that a 57-nucleotide-long region isalternatively retained or spliced out in all of the 10 cell linesstudied (Fig. 4).

The Matrilin-2-specific Antiserum Reacts with Multiple Pro-tein Bands upon Electrophoresis of Unreduced Samples—Inorder to gain information about the relative amount and pre-sumed oligomeric structure of matrilin-2, culture media fromcell lines and extracts from tissues were compared by SDS-

periosteum (po), meniscus, synovial capsule, and ligaments (li) are the most strongly positive areas. Moderate immunostaining of the cartilagematrix (c) is enhanced in the lacunae of hypertrophic chondrocytes (hc). Strong immunoreaction of the developing tendon (te) and staining in thedermis and perimysium can also be observed in the leg. H, in a cross-section of the vertebral column, in addition to the perichondrium (pc),periosteum, and ligaments, note the signal in the dura mater spinalis (dm) and the intense labeling of the epi- and perineurium covering the dorsal(dr) and ventral root fibers as well as the spinal nerve. vb, vertebral body. I, in longitudinal sections of the vertebral column, the annulus fibrosusof the intervertebral discs (ivd) gave the strongest signal for matrilin-2. Tendon (te), ligaments (li), and dermis are also immunoreactive. vb,vertebral body. J, in parallel sections, matrilin-1 was detected in the vertebral bodies (vb), both in the epiphysis and the calcified metaphysis. Bar,0.05 mm (A and C), 0.1 mm (B), 0.2 mm (E and H), 0.266 mm (D), and 0.4 mm (F, G, I, and J).

FIG. 2. In situ hybridization of ma-trilin-2 cRNA to sections of adultmouse organs. Antisense riboprobeswere hybridized to cryostat sections (A–D)or paraffin sections (E–H) of 5-week-oldmice. A–H, light field micrographs, A9–H9are corresponding dark field micrographs.In a longitudinal section of the tail (A andB), mRNA is detected in dermis fibro-blasts, at the base of hair follicle, and intendon (te) cells. A, the stratified, keratin-ized epithelium reflects the light, but isnot truly positive. B, the gene is also ex-pressed in the vertebral body in the peri-chondrium (pc), ligaments (li), and epi-physeal cartilage, especially the zone ofearly hypertrophy (hc) and in cells liningthe bone marrow (bm) cavities. C, thecross-section of uterus shows an overallstrong hybridization signal. Note the ac-cumulation of grains in the perimetrium(pm), mesentery (me), and blood vessels(bv). D, in the heart, the hybridizationsignal increases from the ventricles to-ward the atria and is the strongest in theatrioventricular valves (av), the chordatendinea (ct), and the auricle (not shown).pm, papillary muscle. E, in the obliquesection of trachea (tr) and esophagus (es),the esophageal epithelium, the ciliatedtracheal epithelium, and seromucousglands (gl) in the submucosa and adven-titia give a strong hybridization signal. c,cartilage. F, in a larger magnification, thecartilage ring (c) is weakly positive, incomparison with the signal in the seromu-cous glands (gl). G, in the esophagus, theepithelium is more strongly labeled thanthe subepithelial connective tissue. H, incolon, the mucosa (mu), submucosa, andserosa show a strong signal. me, muscu-laris externa. Scale bar, 0.1 mm (A, B, andF–H), 0.2 mm (C and D), or 0.4 mm (E).

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PAGE and, in part, immunoblotting to the matrilin-2 expressedby transfected cells (Fig. 5C). The specificity of the antiserumwas assessed by comparison of media from 293-EBNA cellsbefore and after transfection with the recombinant plasmidpCEP-Mtr2, encoding the full-length mouse matrilin-2 (Fig.5A). The transfected cells secreted into the culture mediummatrilin-2, which was detected by the antiserum as a group ofantigenic bands in nonreducing SDS-PAGE with apparent Mr

values ranging from 70,000 to 500,000. On the basis of thecalculated Mr 5 104,300 for the nonmodified matrilin-2 mono-mer, faster migrating bands must represent degradation prod-ucts, and the slower ones may correspond to oligomeric forms.We can conclude that the secretory signal peptide of matrilin-2was functionally active in 293-EBNA cells and that matrilin-2can form oligomers stable enough to resist denaturing electro-phoresis if the sample is not reduced. The medium from non-transfected 293-EBNA cells did not show any reactivity withthe antiserum, demonstrating the specificity of this reagent.

The relative amounts of the different forms of matrilin-2,resolved by electrophoresis, differ somewhat between the crudemedia of transfected cells and preparations chromatographi-cally purified from this source (Fig. 5B). The reason for thisdifference is that during purification we removed degradationfragments and enriched slightly for the oligomeric forms. Re-duction of the purified material yielded several closely spaced

bands with apparent Mr values between 100,000 and 130,000.The size heterogeneity was even more apparent when nonre-duced samples from media of a variety of cell lines as well asextracts of skin and uterus were analyzed by immunoblotting(Fig. 5C). All the cell lines tested secreted detectable amountsof matrilin-2, with the exception of the epithelial cell line IEC6.Because the IEC6 cells showed production of matrilin-2 mRNA,we need to assume that the mRNA is translated and/or theprotein is secreted with a very low efficiency in that cell line.While the 293-EBNA cells that had been transfected withpCEP-Mtr2 produced four groups of bands that may representmonomers, dimers, trimers, and tetramers, most cell lines se-creted mainly the smallest and largest components. Extracts ofskin and uterus showed, in addition, a relative abundance ofthe potential trimers. The analysis was, however, complicatedby the presence of discrete differences in the electrophoreticmobility of corresponding matrilin-2 bands between sources(Fig. 5C).

Analysis of Posttranslational Modifications in Matrilin-2—While the presence of matrilin-2 oligomers of variable sizes canbe explained by source-specific differences in assembly, themultiple size of monomers indicated that, in addition to this,posttranslational processing occurs, possibly in a tissue-spe-cific manner. A set of experiments was designed to explore thispossibility (Fig. 6). The most obvious cause for the heterogene-ity would be proteolysis after secretion into the culture me-dium. Samples of medium were, therefore, harvested at differ-ent times after medium change, frozen, and analyzed byimmunoblotting (Fig. 6A). The band patterns of matrilin-2 ob-tained from medium kept with the cells at 37 °C for differentperiods of time show great similarities. With the exception ofdegradation fragments, which are clearly smaller than mono-mers, all components could be observed already after 8 h,indicating that proteolysis in the medium or in the intercellularcompartment of tissues is not the major cause of heterogeneity.The size differences could also be due to a variable substitutionwith glycosaminoglycans or oligosaccharides. To test for thepresence of sulfated glycosaminoglycans, cultures of wild typeand matrilin-2-transfected 293-EBNA cells were, therefore, la-beled with [35S]sulfate, and the media were analyzed by SDS-

FIG. 3. Filter hybridization of cell culture RNA samples. TheRNA samples were isolated from the cell lines indicated at the top. Thefilter was hybridized consecutively with matrilin-2 cDNA (MTR2) and achicken rDNA fragment (28S). For a description of the cell lines, see“Materials and Methods.”

FIG. 4. Analysis of mouse matrilin-2 mRNA heterogeneitywithin the unique region by reverse transcription-polymerasechain reaction. The analysis was performed as described under “Ma-terials and Methods.” Template RNA was isolated from rat chondrosar-coma tissue (RCS) or the mouse and rat cell lines indicated at the top.C, amplification of the longer segment using pCRP12 cDNA clone (4) astemplate. M, pUC12 HaeIII DNA ladder. On the left, sizes of the twoalternative splice products are indicated. At high concentrations, thetwo specific polymerase chain reaction products formed heteroduplexesof lower electrophoretic mobility (hd).

FIG. 5. SDS-PAGE analysis of recombinant matrilin-2 before(A) and after (B) purification and of matrilin-2 in cell media andtissue extracts (C). A, media from 293-EBNA cells either transfectedwith pCEP-Mtr2 (lane 1) or without transfection (lane 2) were appliedto SDS-PAGE without prior reduction. Matrilin-2 was detected by im-munoblotting using an affinity-purified antiserum to matrilin-2. B,purified recombinant matrilin-2 was submitted to SDS-PAGE as in Awithout (2SH) or after (1SH) sample reduction, and the gel wasstained with Coomassie Brilliant Blue. C, media from 293-EBNA cellstransfected with pCEP-Mtr2 and a variety of cell lines as well asextracts from mouse skin and uterus were submitted to SDS-PAGE andimmunoblotting as in A. Nonreduced samples were run on 4–15%polyacrylamide gels; reduced ones were analyzed on 10% polyacryl-amide gels. fn, the position of fibronectin.

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PAGE followed by autofluorography (Fig. 6B). The labelingpatterns seen with the two sets of cultures showed considerablesimilarities, and, most importantly, only minor amounts oflabel were detected in the position of matrilin-2 monomers,which were shown by parallel immunoblotting to be the majormatrilin-2 species in the sample. Further, media from matrilin-2-transfected 293-EBNA cells were digested with the glycos-aminoglycan-degrading enzymes chondroitinase ABC and he-paritinase under standard conditions (Fig. 6C). The treatedsamples appeared unchanged when compared with untreatedsamples by immunoblotting for matrilin-2, which provided fur-ther evidence against a substitution with chondroitin/derma-tan sulfate or heparan sulfate. The potential presence of N-linked oligosaccharides was analyzed in two experiments. Inone approach, parallel cultures of matrilin-2-transfected 293-EBNA cells were treated or not treated with the inhibitortunicamycin (Fig. 6, D and E). SDS-PAGE and immunoblotanalysis of the media showed small but significant increases inthe mobility of matrilin-2 bands, which were, because of thebetter resolution, more easily seen by electrophoresis afterreduction (Fig. 6E). Treatment of purified matrilin-2 with N-glycosidase F gave similarly small, but clearly detectable, de-creases in the apparent Mr as visualized by SDS-PAGE (Fig.6F). Taken together, these results show that the recombinantmatrilin-2 carries N-linked oligosaccharides, but the extent ofthis glycosylation is not sufficient to explain the heterogeneityobserved both with recombinantly expressed matrilin-2 andwith matrilin-2 derived from cell lines and tissues. Neithertunicamycin treatment nor digestion with N-glycosidase F de-creased the heterogeneity of recombinant matrilin-2, a findingthat points to another underlying cause.

Electron Microscopy Reveals That Matrilin-2 Occurs as aMixture of Monomers and Oligomers—Purified, recombinantmatrilin-2 (Fig. 5B) was negatively stained with uranyl for-

mate to produce high resolution images by electron microscopy.Fields of stained molecules showed particles of heterogenoussize (Fig. 7, top), and close examination of single particlesrevealed that all species from monomers to tetramers werepresent in the sample. At high magnification it was seen that,irrespective of the number of subunits within a molecule, allsubunits are joined at a single point (Fig. 7, bottom), whichpresumably represents the coiled-coil a-helix assembled fromthe C-terminal domains. In most cases, the subunit is seen asa looped structure with a more heavily stained hole in themiddle and frequently, but not always, carrying most mass inthe periphery. The average diameter of the loops was 8 nm.Occasionally particles were seen where the loop was openedinto a flexible rod, better representing the tandem array ofdomains making up the subunits.

The Protein Forms an Extracellular Filamentous Network inCell Culture—In order to study the extracellular assemblyforms of matrilin-2, the pericellular matrix of cultured primarysmooth muscle cells from rat aorta was analyzed in immuno-fluorescence microscopy with specific antibodies against matri-lin-2 (Fig. 8). Matrilin-2 was detected in an extensive, branchedfibrillar network. The network may be connected to the cellsurface, but in experiments where the cells were permeabilized(Fig. 8B), and thereby better outlined through the staining ofthe intracellular pool of immunoreactive matrilin-2, it wasclear that the fibrils were not limited to the cell surface butextended over and beyond cells.

DISCUSSION

The Matrilin-2 Gene Is Transcribed in Fibroblasts, Myocytes,and Epithelial Cells, but the Protein Is Transported to Connec-tive Tissue Structures—During our previous work, matrilin-2mRNA was found in skin of the tail, calvaria, heart, uterus, andbrain (4). From the wide distribution of the transcript, expres-sion of the gene in cell types common to many organs waspresumed. In situ hybridization and Northern blotting datarepresented here confirmed gene expression in connective tis-sue cells but extended the expression pattern to muscle andepithelial cells. Most of the expressor cell types are of mesoder-mal origin, but the epithelia of trachea, esophagus, and intes-tine, which showed strong in situ hybridization signals, developfrom endoderm. Expression of the matrilin-2 gene in cells of

FIG. 6. Analysis of the heterogeneity of matrilin-2 by SDS-PAGE. A, accumulation of matrilin-2 in cell culture media. Sampleswere harvested from 293-EBNA cells transfected with pCEP-Mtr2 8,24, and 48 h after medium change. Aliquots were applied to a 4–15%SDS-PAGE without prior reduction. Matrilin-2 was detected by immu-noblotting using an affinity-purified antiserum. B, electrophoretic anal-ysis of sulfate-labeled products secreted into the medium. 293-EBNAcells either transfected with pCEP-Mtr2 (lane 2) or without transfection(lane 3) were cultured in the presence of 50 mCi/ml [35S]sulfate for 48 h,and aliquots of the media were applied to a 4–15% SDS-PAGE withoutprior reduction. The radioactive macromolecules were detected byautofluorography. In parallel, a lane containing medium from thetransfected cells was blotted and reacted with the matrilin-2 antiserum(lane 1). C, media from matrilin-2-transfected cells were digested withheparitinase (lane 1), not digested (lane 2), or digested with chondroiti-nase ABC (lane 3). The samples were applied to gel electrophoresis anddeveloped by immunoblotting as in A. D and E, 293-EBNA cells trans-fected with pCEP-Mtr2 were cultured in the presence (1) or absence (2)of 0.5 mg/ml tunicamycin, and aliquots of media were applied to either4–15% SDS-PAGE without prior reduction (D) or 6% SDS-PAGE afterreduction (E). Matrilin-2 was detected in the samples by immunoblotanalysis. F, purified recombinant matrilin-2 was digested (1) or notdigested (2) with N-glycosidase F, applied to a 4–15% SDS-PAGE, andstained with silver nitrate.

FIG. 7. Negative stain electron microscopy of purified recom-binant matrilin-2. An overview (top) and selected particles at highermagnification (bottom) show the heterogeneity in oligomerization andillustrate the presence of subunits in a looped (particles 1, 2, 4, and 6from the left) or open (particles 3 and 5 from the left) conformation. Thebar corresponds to 100 nm for the overview (top) and 25 nm for theenlarged single molecules (bottom).

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various developmental lineages predicts a complex mechanismfor regulation of gene expression.

In some cases, synthesis of the matrilin-2 mRNA was foundin epithelial cells, but deposition of the protein was observedonly in subepithelial connective tissue. It is likely that thematrilin-2 mRNA is translated in epithelial cells and that theprotein is specifically transported to the subepithelial regions.It is known that some components of basement membranes aremade by epithelial cells; others are contributions from theunderlying connective tissue fibroblasts. Nidogen is exclusivelymade and the a1 and a2 chains of collagen IV are predomi-nantly produced by dermal fibroblasts, but all three chains oflaminin-1 can be expressed by keratinocytes, especially at thebeginning of coculturing (20). In the case of matrilin-2, thepossibility cannot be excluded that epithelial cells synthesizemRNA, but translation or protein transport is blocked. Theabsence of matrilin-2 from media of the IEC6 intestinal epithe-lial cell line (Fig. 5C) supports this alternative, but this cell linemight also not be representative for the differentiation state ofepithelial cells in vivo.

Matrilin-2 Displays an Unusual Extent of Structural Heter-ogeneity—In preparations of recombinant matrilin-2, at leastthree bands of reduced monomers can be resolved by SDS-PAGE (Fig. 6E). The size differences observed between matri-lin-2 from different sources appear too large to be explained bythese three bands occurring in variable proportions. In studiesusing the recombinant matrilin-2, we have excluded extracel-lular proteolysis and substitution with glycosaminoglycans orN-linked oligosaccharides as the major source of heterogeneity.Since the recombinant matrilin-2 is derived from cloned cDNA,alternative splicing may also be excluded. Remaining possibil-ities are unconventional forms of substitution and/or process-ing by intracellular or cell surface-bound proteases during bio-synthesis and secretion. Further studies will be needed toevaluate these alternatives and to determine if the processinghas functional consequences.

SDS-PAGE revealed also a second level of heterogeneity,with bands corresponding to various oligomeric species beingpresent in recombinant matrilin-2 (Fig. 5). This cannot beexplained by imperfect assembly due to overexpression in thetransfected 293-EBNA cells, since similarly variable oligomer-ization is seen in tissue extracts and in media from a variety ofcell lines (Fig. 5C). Different assembly forms representing all

species from monomers to tetramers are seen in electron mi-croscopy of samples purified under nondenaturing conditions(Fig. 7). This proves that the occurrence of multiple bands inSDS-PAGE is not due to incomplete closure of interchain di-sulfide bridges followed by dissociation of the coiled-coil upontreatment with SDS, but either that coiled-coil a-helices withvarying numbers of protein strands are formed or that matri-lin-2 subunits are specifically proteolytically cleaved at a siteclose to the coiled-coil region before or around secretion. Panand Beck (6) recently investigated the oligomerization of asynthetic peptide corresponding to the coiled-coil domain ofmatrilin-2. By means of chemical cross-linking, they found thata trimer was the preferred species, but even at high cross-linking reagent concentrations, at which a corresponding ma-trilin-1-related peptide exclusively shows a single band corre-sponding to a trimeric state (21), the matrilin-2 peptide showednearly equal amounts of material running in the position ofmonomers, dimers, and trimers (6). Under these conditions,higher molecular mass bands, although of lower concentra-tions, which can be interpreted to represent tetramers andpentamers, were also found. The multiplicity of oligomers couldnot be abolished by increasing the ionic strength as it wasfound for the matrilin-1 peptide (21). Although these authorsargue that the monomeric and dimeric states observed uponSDS-PAGE might be due to the limitations of the cross-linkingapproach, they could not rule out that indeed different oli-gomerization states for the matrilin-2 related peptide are pos-sible. Studies of matrilin-1 derived from tissue sources showthat homooligomerization of the naturally occurring proteinleads to the formation of trimers as the single predominantspecies (18). Recent work has, however, demonstrated restric-tions in the stringency of coiled-coil formation among matrilinsin showing that a single point mutation in the coiled-coil do-main of matrilin-1 may cause it to form tetramers instead oftrimers (22) and that matrilin-1 occurs in vivo not only as ahomotrimer but also in a heterotetramer together with matri-lin-3 subunits (23). In the case of matrilin-2, it could be thatthis lack of stringency leads to the protein occurring in vivo asa mixed set of monomers and oligomers. This heterogeneitymay have functional consequences, since ligand binding siteswill occur in variable copy number within a single molecule,and differences in oligomerization state may, because of coop-erativity, lead to differences in affinity for macromolecularligands that may bind to more than one subunit within a singlematrilin-2 molecule. The possibility cannot be excluded thatthe oligomers detected by immunoblot are heterooligomersformed between matrilin-2 and another matrilin. A candidatewould be matrilin-4, which is expressed outside cartilage asshown by Northern blots that give signals in lung, liver, brain,sternum, kidney, and heart (5).

Evidence for Self-interaction of vWFA Domains of Matrilin-2and Its Potential Role in Fibrillar Network Formation in CellCulture—In electron microscopy using negative stain, the ma-trilin-2 subunits are most often seen as loop structures with adiameter of 8 nm, but in a smaller fraction of the particles theyare seen to open into a flexible rod (Fig. 7). Based on x-raycrystallographic data, we may assume a diameter of 3.6 nm forvWFA domains and 2.1 nm for EGF-like domains. In a tandemarray, the two A domains together with the 10 EGF-like do-mains would have a length of about 28 nm. A circle with adiameter of 8 nm has a circumference of 25 nm, and the meas-urements are, therefore, compatible with a model of the matri-lin-2 subunit where a loop is formed through interactions be-tween the two A domains. In earlier studies of matrilin-1, wesimilarly observed a compact structure of the subunits (18).Domain interactions within the subunit had to be assumed to

FIG. 8. Immunofluorescence microscopy of matrilin-2-contain-ing assemblies in the pericellular matrix of cultured primaryrat aorta smooth muscle cells. Confluent cell layers were fixed in 2%paraformaldehyde, and matrilin-2 was detected with affinity-purifiedantibodies without (A) and with (B) permeabilization with 10% NonidetP-40. An extensive network of matrilin-2-containing filaments is seenextracellularly, and after permeabilization an intracellular precursorpool is also detected. Bar, 20 mm.

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explain why the length determined from electron micrographswas considerably smaller than that expected from the dimen-sions of the domains in tandem array. Since the A-domains inmatrilin-1 are connected by a single EGF-like domain, sub-structures within the subunit could not be resolved, while inthe matrilin-2 subunit, where the A-domains are connected by10 EGF-like domains, a loop around a heavily stained holecould be seen. For sterical reasons, it is most likely that theloop is formed by the EGF-like domains, which are held to-gether through an interaction between the two A-domains.Indeed, structures indicating self-interactions between A-domains have been observed by electron microscopy of vonWillebrand factor (24) and of the N-terminal globule of the a3chain of type VI collagen (25). Further, by x-ray crystallogra-phy of von Willebrand factor A1-domain a contact surface wasdetected between A1-domain pairs, suggesting a hypotheticalmechanism for the regulation of protein assembly and heterol-ogous ligand binding mediated by homophilic interactions oftype A-domains (26). vWFA domains in those proteins havealso been shown to be involved in other interactions (25, 27).

By immunofluorescence microscopy of the matrix formed bycultured smooth muscle cells (Fig. 8), we could show that ma-trilin-2 molecules assemble into an extracellular fibrillar net-work, where each fibril may have a length of several cell diam-eters and often divides into smaller branches. In similarstudies, matrilin-1 was found in chondrocyte cultures in closeassociation with collagen II fibers (28), and a filamentous net-work, independent of collagen fibers, was also observed whenmatrilin-1 was overexpressed using a retroviral system (29).Matrilin-1 constructs, in which the vWFA1 domain had beendeleted, assembled into trimers but could not form filamentousstructures, thereby implicating the vWFA1 domain as beinginvolved in the polymerization reaction leading to fibril forma-tion. In analogy, it is likely that the vWFA domains of matri-lin-2 may interact with each other and that filaments may beformed by this interaction. We do not know at present the exactmolecular composition of the matrilin-2-positive extracellularfilaments, but we are pursuing the study of such interactions ofmatrilin-2 with itself and with other extracellular macromole-cules that may form the basis for fibril formation.

Acknowledgments—We are indebted to Drs. G. K. Owens, J. H.Kimura, L. Sorokin, R. Hallmann, N. E. Fusenig, F. Michaelsen, and J.Karolat for providing the cell lines and to L. Modis, K. Addicks, and K.Beck for valuable discussion. We also thank I. Fekete for technicalassistance and A. Borka and M. Toth for artwork.

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Kiss, Mats Paulsson and Ferenc DeákDorothea Piecha, Selen Muratoglu, Matthias Mörgelin, Nik Hauser, Daniel Studer, Ibolya

Cells and Forms Fibrillar NetworksMatrilin-2, a Large, Oligomeric Matrix Protein, Is Expressed by a Great Variety of

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