Undulin, an Extracellular Matrix Glycoprotein Associated with Collagen Fibrils*

Undulin, a novel noncollagenous extracellular matrix protein, was isolated from skin and placenta. In polyacrylamide gels most of the unreduced protein migrates with Mr above 1,000,000 yielding bands A (Mr 270,000), B1 (Mr 190,000), and B2 (Mr 180,000) after reduction. Undulin is biochemically and immunochemically distinct from other previously characterized large matrix glycoproteins. Immunoblotting using monoclonal antibodies suggests that bands A and B are closely related. Electron microscopy reveals undulin as structures consisting of an approximately 80-nm-long-tail with a nodule on one end and with one or two shorter arms on the other. Ultrastructurally immunolabeled undulin is found mainly between densely packed mature collagen fibrils. Indirect immunofluorescence shows bundles of uniform wavy fibers in dense connective tissues superimposable on a subpopulation of type I collagen structures. This suggests that undulin serves a specific yet unknown function in the supramolecular organization of collagen fibrils in soft tissues.

As a major determinator of cellular gene expression and differentiation the extracellular matrix (ECM)' is of fundamental importance for the coordinate growth and maintenance of multicellular organisms (Hay, 1984;Thiery et al., 1985). With the advent of refined biochemical and ultrastructural methods a perplexing variety of matrix molecules equipped with unforeseen biological activities has been disclosed (Schuppan and Hahn, 1987). Rapid progress has been made with the characterization of at least 12 types of collagen (Miller and Gay, 1987;Gordon et al., 1987), various proteoglycans (Heinegard and Paulsson, 1984;Poole, 1986), and glycoproteins (Hakamori et al., 1984). Each of these molecular species bears specific domains evidently capable of performing multiple structural or signal-inducing functions. In view of their high potential for interaction, much interest has been focused on the large ECM glycoproteins fibronectin, laminin, thrombospondin, and tenascin. Fibronectin (M, 540,000) is a constituent of plasma as well as the interstitial ECM. It is involved in blood coagulation, wound healing, and chemotaxis.
It mediates the interaction of cells with other matrix components such as fibrillar collagen and heparin (Hynes, 1985;Yamada et al., 1985). cDNA cloning of fibronectin mRNA demonstrated three types of internal homologies (Kornblihtt et al., 1986) and a tripeptide sequence (Arg-Gly-Asp), which is recognized by the cellular fibronectin receptor and has later been found to be a common feature of many ECM molecules (Ruoslahti et al., 1987).
Laminin (M, 900,000) is a cross-shaped molecule unique to basement membranes. The glycoprotein, which links cells to the scaffold of type IV collagen and interacts with heparan sulfate proteoglycan and entactinlnidogen, has received broad attention as a promoter of epithelial/endothelial differentiation, neurite outgrowth, and as a target to which metastasizing cells preferentially attach (Martin and Timpl, 1987). Recently, the complete amino acid sequence of its three constituent chains has become available (Sasaki et al., 1988). Laminin harbors at least two domains which mediate binding to cell surface receptors (Aumailley et al., 1987). One of these activities resides in a pentapeptide sequence close to the central part of the molecule (Graf et al., 1987).
Thrombospondin (Mr 420,000) composed of three identical subunits, is produced by various cell types and interacts with fibronectin, laminin, type V collagen, and fibrinogen (Lawler, 1986). Its sequence shows only limited homology with that of the other large ECM glycoproteins (Lawler and Hynes, 1986). Tenascin (Chiquet-Ehrismann et al., 1986), which has a six-armed structure of M, l,OOO,OOO, is identical or similar to glioma mesenchymal extracellular matrix antigen (Bourdon et al., 1983), myotendinous antigen (Chiquet andFarmbrough, 1984), hexabrachion (Erickson and Inglesias, 1984), Jl (Kruse et al., 1985), and cytotactin (Grumet et al., 1985). It is expressed in regions where epithelial/mesenchymal interactions take place during embryogenesis, e.g. around limb buds (Chiquet and Farmbrough, 1984) and budding kidney tubules (Aufderheide et al., 1987), and is reexpressed in the mesenthyme bordering invasive carcinomas (Mackie et al., 1987). A major part of the protein sequence of tenascin/cytotactin has been recently determined (Gulcher et al., 1989;Jones et al., 1988;Pearson et al., 1989) showing clear homologies to domains in fibronectin and demonstrating that the differentsized bands observed in polyacrylamide gels are derived from differential splicing. Here we report on the isolation and characterization of a novel ECM glycoprotein which is associated with mature collagen fibrils. Light and electron microscopical studies with the aid of monoclonal antibodies revealed this glycoprotein, which is produced by several mesenchymal cell lines, as a unique and abundant component of certain dense interstitial connective tissues. We propose for the novel protein the name "undulin" which is derived from the latin "undula" (little wave) and describes its highly characteristic light microscopical pattern of uniform wavy fibers often organized into regular fiber bundles.
Unbound material (type I and III collagen) was removed and the proteins bound to the resin were eluted with a gradient of 0.02-0.5 M NaCl. Upon elution undulin, which was about 80% pure, was displaced from the column after type I and III procollagen (Fig. 2) were purified from human placenta and characterized as described previously (Becker et al., 1986aSchuppan et al., 1985Schuppan et al., , 1986a and laminin fragment Pl as described elsewhere (Becker et al., 1986a, 198615;Schuppan et al., 1985Schuppan et al., , 1986aSchuppan et al., , 1986b. The antibodies bound to the undulin column were finally eluted using 3 M KSCN in PBS, 0.05% Tween 20 and dialyzed against PBS, 0.05% Tween 20. The production of antisera and affinity-purified antibodies against monkey type I and III procollagen; human type IV (7-S long, triple helical, and NC1 domains), V, and VI collagen, and laminin fragment Pl and their characterization as monospecific by sensitive radioimmunoassays have been described before (Becker et al., 1986aSchuppan et al., 1985Schuppan et al., , 1986aSchuppan et al., , 1986b The same amount of antigen in incomplete adjuvant was administered after 4 weeks and given daily for the last 4 days before the fusion was carried out. Hybridomas were produced, subcloned, and propagated in ascites fluid as described by Kohler and Milstein (1975). Clone selection was done with the aid of indirect immunofluorescence and enzyme-linked immunosorbent assay (Engvall and Perlman, 1971). Antibody clones 11 BI (IgGl), 15 III IgG2a), and 15 V (IgCBb) were used in the present study. Radioimmunoassa.ys-Iodine-125 was incorporated into all antigens except for the NC1 domain of type IV collagen by the chloramine-T method. The NC1 domain had to be labeled with the Bolton-Hunter reagent (Schuppan et al., 1985. Undulin was dissolved in 50 mM Tris-HCl, 2% SDS, pH 8.3, or 50 mM Tris-HCl, 6 M suanidine. For radiobinding assays (Schuppan et al., 1985) serial dilutions of antibodies in 0.2 ml of 0.25% carrier rabbit/goat serum in PBS, 0.05% Tween 20 were incubated for 4 h with the radiolabeled antigen (20,000 cpm, l-5 ng of protein in 0.2 ml), followed by precipitation of immune complexes with a pretested amount of second antiserum (usually goat serum against rabbit IgG). Analytical Methods and Immunoblotting-Proteins were analyzed by SDS-PAGE at concentrations of 2-20 Kg/slot (Laemmli, 1970). Antigens run on gels were blotted to nitrocellulose, and, after blocking unspecific binding sites with 25 mM Tris-HCl, 0.05% Tween 20, pH 7.4, containing 2% delipidated milk powder, the blots were developed essentially as outlined by Dziadek et al. (1983) using primary antibodies at l-6 pg/ml, goat anti-rabbit IgG, or rabbit anti-goat IgG coupled to peroxidase (Calbiochem, FRG) and a mixture of o-catechol and p-phenylenediamine as substrates. Alternatively, antigens were detected with alkaline phosphatase-linked secondary antibodies, nitro blue tetrazolium, and o-chloroindolyl phosphate as indicator (Blake et al., 1984).
Peptide mapping of undulin bands A and B was performed according to the method of Cleveland et al. (1977). Briefly, reduced undulin was run on a 6% acrylamide gel and the bands were excised and layered on top of a 13% acrylamide gel with a 4% stacking gel. After addition of Staphylococcus protease V8 (2 rg/lO h1 sample buffer, Sigma type XVII) the gel was run at 100 V until the dye front reached the lower third of the stacking gel. After the gel was left to incubate at room temperature for 1 h the electrophoresis was completed at 200 V. Gels were either silver-stained (Schoenle et al., 1984)  were shadowed with platinum-carbon at an angle of about 5" and with carbon at 90" in a Balzers BAE 400 coating unit (Furthmayr and Madri, 1982 Immunoelectron Microscopy-Freshly collected biopsies of human skin and oral mucosa were prepared for immunoelectron microscopy after ultracryomicrotomy as described (Gelderblom et al., 1985). Briefly, tissue cubes were fixed in dimethyl suberimidate, pH 9.5, and infiltrated with 70% sucrose. Ultrathin sections were cut in a Reichert-Jung FC-4 cryostat at -90 "C and transferred to Formvar-coated, carbon-reinforced hexagonal copper grids. Sections were incubated with the desired polyclonal or a mixture of monoclonal antibodies 11 BI, 15 III, and 15 V at 150-250 @g/ml for 10 min and then with goat anti-rabbit or rabbit anti-mouse gold conjugate (5-nm particle size, Janssen, Belgium) diluted 1:60, for 20 min. The specimens were postfixed with 2% glutaraldehyde and stained with 2% uranyl acetate. For control purposes equivalent amounts of rabbit or mouse nonimmune IgG were used in place of the primary antibodies. Immunostaining was observed using a Zeiss 10A electron microscope.

Isolation and Characterization
of Undulin-As illustrated in Fig. 1 undulin was isolated from monkey skin and human placenta by extraction with neutral buffer and salt fractionation employing a procedure similar to that used to isolate interstitial collagens and procollagens (Sage and Bornstein, 1982). Material precipitating with type I collagen and procollagen contained the bulk of undulin mainly in a form insoluble in low salt buffers. When chromatographed on DEAE-cellulose, a distinct protein peak was eluted with a salt gradient at 0.2 M NaCl (Fig. 2, fraction d). This peak was only observed when strict protease inhibition was implemented during the initial purification.
SDS-gel electrophoresis (Fig. 3A) indicated that the less acidic fractions eluting from DEAE-cellulose (b and c in Fig. 2) contained type I and III procollagen. Electrophoresis of fraction d, however, displayed a hitherto unknown pattern. Most of the unreduced material in fraction d did not penetrate the stacking gel. After reduction, characteristic bands of M, 270,000 190,000, and 180,000 relative to collagen and fibronectin standards appeared. In the following these components of undulin are designated as the A, Bl, and B2 bands, respectively. The B band doublet migrated with a mobility higher than that of reduced fibronectin and with a lower mobility than that of type I and III procollagen.
In four different preparations undulin consistently cochromatographed with approximately 10% of type I collagen and traces of type III collagen and procollagen.
This was unexpected, since type I collagen alone does not bind to DEAE-cellulose under the conditions used. Separation of collagen, fibronectin, and material of lower molecular weight was achieved by chromatography on a column of Sepharose S-500 in buffer containing 6 M guanidinium. Undulin eluted from the column as a second sharp peak in the region of mouse tumor laminin (Mr 900,000, not shown). Undulin thus purified and subsequently reduced showed the expected pattern with the B bands less clearly separated than in preparations of native material. In addition, some faster-migrating bands designated A' and B' (Fig. 3B), possibly generated due to residual proteolytic activity, appeared. The yield of pure undulin from newborn monkey skin and human placenta was 50-100 mg/kg wet tissue weight. Amino acid analysis of purified undulin revealed a high proportion of hydrophobic and acidic residues, whereas hydroxyproline and hydroxylysine, which are characteristic for collagens, were absent. The overall composition is distinct from that of fibronectin or laminin (Table I) and of thrombospondin (Coligan and Slayter, 1984, data not shown). Carbohydrate analysis showed that undulin is a glycoprotein with N-Asp (GlcNAc, Man) and Ser/Thr (GalNAc, Glc)-linked oligosaccharides.
As demonstrated in Table II the degree of glycosylation (2% relative to protein) is significantly lower than that reported for tissue fibronectin (-4%) and laminin (-12%).

Undulin
Is Unrelated to Other ECM Antigens-In order to test whether undulin is related to other known connective tissue components, a rabbit antiserum was produced against the monkey protein. The antiserum was passed over a wide spectrum of immobilized human/monkey ECM antigens (type  I and III collagen and procollagen, type IV (7-S long, triple helical, and NC1 domains), V and VI collagen, fibronectin and laminin fragment Pl). Monospecific antibodies were obtained by a final chromatography on an undulin column. There was no significant binding of the undulin antibodies to the radiolabeled ECM antigens mentioned above whereas the antibodies did bind to radiolabeled undulin with high affinity (Fig. 4A). Likewise, radiolabeled undulin did not bind significantly to high titer antibodies against all of the abovementioned antigens (Fig. 4B). These data suggest that undulin is immunologically unrelated to these collagens and ECM glycoproteins.
Up to this point there was no sound evidence that all of the three bands attributed to undulin were indeed the antigens reactive to the polyclonal undulin antibodies. As illustrated in the immunoblot of a crude monkey skin extract in Fig. 5A, bands A, Bl, and B2 were recognized by the antibodies. Without reduction high molecular weight material not penetrating the stacking gel or just entering the running gel was seen. Antibodies to human and monkey type I and III collagen and procollagen; type IV (7-S, triple helical, and NC1 domains), V, and VI collagen; fibronectin; laminin fragment Pl; and chicken tenascin failed to react with the bands considered characteristic for undulin. Furthermore, undulin antibodies collagen and monkey type I procollagen was less than 5% at an antibody concentration of 10-l and therefore is not shown. R, binding of "'I-labeled monkey undulin to polyclonal antibodies against ECM molecules. 0, antiundulin (n-Un); A, anti-type VI collagen (o-VI); 0, anti-monkey type I procollagen (tu-PIP). Binding to antibodies against human type I, III, IV (7-S long, triple helical, and NC1 domains), and V collagen, laminin fragment Pl, and the bovine amino-terminal type III procollagen propeptide, which was below 5% at an antibody concentration of lo-', is not shown. did not react with the antigens mentioned (not shown). This constitutes further evidence that undulin is unrelated to these ECM proteins and that A, Bl, and B2 are related to one parent molecule. Bands A, Bl, and B2 of Undulin Are Closely Related-Three monoclonal antibodies were produced using monkey undulin as the immunogen. When a human placental undulin preparation of about 50% purity (prior to molecular exclusion chromatography) was subjected to SDS-gel electrophoresis, transferred to nitrocellulose, and probed with these antibodies a pattern essentially identical to that observed with polyclonal A, (lanes 1 and 2) a tissue extract from monkey skin with less than 5% undulin was run in SDS-PAGE (6% acrylamide) without (-) and after (+) reduction.
(lanes 3 and 4) the same material as in (1) and (2)  antibodies was obtained, mainly A, Bl, and B2 bands and some faint higher as well as lower molecular weight material reacted with the antibodies (Fig. 5B). This indicates that A, Bl, and B2 belong to one molecular species and that either the three bands have major immunogenic epitopes in common or, more probably, Bl and B2 are derived from A by pretranslational or posttranslational processing.
Additional evidence for the relatedness of A and Bl/B2 was obtained from peptide maps of the isolated bands, which clearly demonstrated that Bl/B2 contain a major subset of peptides also found in A. Interestingly, when probed with a monoclonal antibody, the peptide map of A displayed an additional doublet around M, 55,000 and material of higher molecular weight strongly reactive with the antibody suggesting that A is derived from a larger differentially spliced section of the undulin gene (Fig. 6).
Composition of Undulin from Several Tissues-In order to investigate the composition of undulin from different tissues 20 mg each of human placenta, uterus, liver, and large intestine were extracted directly with nonreducing or reducing SDS sample buffer, run on SDS gels, and transferred to nitrocellulose. As demonstrated in Fig. 7A polyclonal and monoclonal antibodies equally detected material in the position of A, Bl/B2, and some higher as well as lower molecular weight material. This implies that 1) undulin is a ubiquitous component of connective tissue and 2) undulins from different tissues are identical or at least similar.

Cells Producing
Undulin in Culture-The synthesis of undulin by several mesenchymal cell lines was shown with the aid of indirect immunofluorescence. Whereas only less than 10% of preconfluent human foreskin fibroblasts and diploid lung fibroblasts (WI-38, MRC-5) exhibited undulin-positive intracellular granules, cell lines from a rhabdomyosarcoma (RD) and an osteosarcoma (MG-63) consistently displayed strong immunoreactivity in more than 50% of preconfluent cells (Fig. 8). To investigate the composition of undulin produced by cells in uitro, culture medium from RD cells was subjected to immunoprecipitation with monoclonal undulin antibodies followed by SDS-gel electrophoresis and immunoblotting (Fig. 7B)  Procedures." A, extracts from human placenta (lanes 1 and 5), uterus (lanes 2 and 6), liver (lanes 3 and 7), and large intestine (Innes 4 and 8) without (lanes 1-4) and after (lanes 5-6) reduction of disulfides. 5 /*l of extract, equivalent to 0.5 mg of tissue, were applied to each lane. Note the presence of faint bands slightly above and below the A band as well as material of M, 160,000, 140,000, and 120,000 (small arrows) in all preparations. B, immunoprecipitate from RD cell culture medium representing one sixth of the material from preconfluent 75-cm' culture flasks without (-) and after (+) reduction of disulfides. tissues and 2) the bands of lower molecular weight after extraction from tissues (Fig. 7A) are either derived from degradation during the isolation or result from successive processing of the matrix form in uiuo.

Undulin
Has a Unique Molecular Shape-When examined by rotary-shadowing electron microscopy, putatively monomeric forms of undulin could be visualized (Fig. 9A). Although only preparations of the molecule denatured in 6 M guanidine were studied some distinct features could be observed. The structures consisted of an approximately 80-nm-long threadlike segment, terminating at one end in a nodule with a diameter of -9 nm and linked at the other end to one or two -45-nm-long arms. Sometimes the arms appeared to be stud-  (A) and di/trimers (R). Bar, 100 nm. An approximate 80-nm long threadlike segment is terminated at one end by a nodule with a diameter of approximately 9 nm and linked at the other end to one or two -45-nm-long arms, which often appear to bear two or three smaller nodules (best visualized in A). For further details refer to "Results" and "Experimental Procedures." ded with two to three smaller nodules. Many forms observed in our preparations were evidently aggregates of these monomers like the presumed dimers and trimers shown in Fig. 9B and other higher order aggregates (not shown). The structures seen did not resemble ECM molecules previously characterized by rotary shadowing including fibronectin, tenascin/ cytotactin/hexabrachion, laminin, or collagens/procollagens. A superficial similarity to rotary-shadowed thrombospondin, a tripartite molecule composed of three identical arms each terminating in a globule with a diameter of 8-12 nm (Coligan and Slayter, 1984;Lawler et al., 1985), could be dismissed on closer inspection: 1) undulin was composed of a long arm and one or two shorter arms compared to the uniform arms in thrombospondin and more importantly 2) the arms in undulin were significantly longer (approximately 45 and 80 nm) than those observed in thrombospondin (approximately 29-30 nm in a contracted state and 38 nm in an extended state; Lawler et al., 1985). Significant amounts of other species were not observed in our preparations.
Unfortunately the denaturing treatment of undulin during its purification from tissue invariably led to increased numbers of aggregates as evidenced by SDS-gel electrophoresis even after reduction of disulfides. Thus, visualization of the native protein, e.g. isolated from cell culture, could aid in defining its molecular architecture more conclusively.
Undulin Displays a Distinctive Distribution in the ECM-Since information on the tissue distribution of an assumed novel ECM protein could yield important clues about its potential biological role, we performed indirect immunofluorescence on sections of several human organs. Staining of skin and oral mucosa revealed a highly characteristic, previously unobserved pattern of uniform undulating fibers organized in bundles. The bundles were found extending throughout parts of the dense interstitium, regardless of whether polyclonal or monoclonal antibodies were used to visualize them ( Fig. 10, a, and b). These fibers were found in areas rich in fibrillar collagen, such as the dermal corium or as an outer sheath around blood vessels, glands, and nerve fibers. Cells, basement membranes, and bone were not stained by the antibodies.
In the liver the densely packed fiber bundles of the portal ECM contrasted to the sparsely outlined perisinusoidal space (Fig. 10, c and d). Whereas the medial and intimal layers of small arteries were unstained, the media of larger arteries showed an amorphous fluorescence for undulin bordered by the characteristic undulating fiber bundles in the vascular adventitia (Fig. 10, e and f). This unique restricted pattern of distribution has not been found for other ECM proteins including thrombospondin which contrary to undulin is concentrated at the endothelial aspect of blood vessels and in basement membranes such as that separating epidermis from dermis (Wright et al., 1985).
Double-staining Immunofluorescence-Tissue sections were stained sequentially with antibodies against undulin and other ECM components. As shown in Fig. 11, a and b, tenascin and undulin in placenta, for example, were distributed exclusively of each other, i.e undulin is expressed where tenascin is absent and vice versa. This exclusive distribution was found in all tissues thus far examined including liver, skin, oral mucosa, and gut. In contrast, undulin was codistributed with a subgroup of type I collagen fibers in the dense fibrillar ECM of the dermis, at the periphery of vascular structures, nerve fibers, glands, and of smooth muscle bordering the interstitium, i.e. structures requiring a pliable sheath of collagen fibers (Fig. 11, e and f). Undulin was only partly codistributed with fibronectin and was absent, e.g. in loose connective tissue and in the walls of blood capillaries (Fig. 11, c and d).
Undulin Is Associated with Collagen Fibrils-To explore further the association of undulin with fibrillar collagen, we carried out immunoelectron microscopy with polyclonal or monoclonal antibodies on ultrathin cryostat sections of human dermis and oral mucosa. Using gold labeling, we detected the protein almost exclusively on the surface of mature (diameter above 60 nm) collagen fibrils (Fig. 12). Label occurred discontinuously along the fibril axis and appeared to be concentrated where the collagen fibrils were densely packed. This ultrastructural distribution is quite distinct from that described for type I and III collagen and procollagen, which are labeled at regular intervals along the fibril axis (Fleischmajer et al., 1981), type VI collagen, which forms long microfibrils between individual collagen fibrils (Von der Mark et al., 1984) and fibronectin, which is found predominantly on nonfibrillar structures or deposited in spaces between loosely packed collagen fibrils . DISCUSSION A novel ECM glycoprotein, named undulin due to its characteristic light microscopical pattern of distribution in interstitial tissues, is described. Evidence that undulin is a distinct protein of the ECM is based on several observations: (1) undulin is immunologically unrelated to other macromolecules of the interstitial ECM such as several collagens/procollagens, fibronectin, laminin, and tenascin/cytotactin; (2) amino acid and carbohydrate analyses showed that this component is not a collagen, but a glycoprotein distinct from the other known ECM glycoproteins; (3) rotary shadowing electron microscopy visualized undulin as a previously unknown molecular species; (4) studies with immunofluorescence microscopy indicated that its distribution in tissues is unique when compared to that of other ECM components; (5) immunoelectron microscopy revealed its close association with collagen fibrils at sites where these fibrils are tightly packed, FIB:. 11. Undulin shows limited codistribution with type I collagen and a complementary distribution to tenascin.
Douhle-staining immunofluorescence on cryosections of human placenta (n and b) and uterus (c-f). Bars, 40 pm. a, rabbit antitenascin shows a distribution completely complementary to that of monoclonal antiundulin in h. Arterioles (arrou:heads) and some vascular structures are positive fbr tenascin. c, monoclonal antifibronectin strongly labels a capillary (arrow) and a vascular structure (arrowhead). d, rabbit antiundulin is partly codistributed with f'ibronectin, but is absent l'rom the areas marked by arroux A. arteriole. c, rabbit anti-type 1 collagen stains fibers throughout the uterus and the pattern obtained with monoclonal antiundulin (f) corresponds at specific sites, such as the sheaths surrounding an arteriole (A) and smooth muscle fibers (M). but not at others such as the interstitium between individual muscle cells.
an ultrastructural distribution not yet described for any other ECM molecule.
Although undulin is clearly a distinct glycoprotein of the ECM, it bears some similarities to other ECM macromolecules, particularly tenascin. Both molecules display a molecular weight above l,OOO,OOO on SDS gels when unreduced.
After reduction of disulfides undulin shows bands of M, 270,000, 190,000, and 180,000, whereas tenascin/tenascin-like molecules show two to three bands between 150,000 and 260,000 depending on the species, its developmental stage, and the tissue of origin (Aufderheide and Ekblom, 1988;Bourdon et al., 1983;Chiquet and Farmbrough, 1984;Chiquet-Ehrismann, 1986;Grumet et al., 1985;Hoffman et al., 1988;Kruse et al., 1985). It has recently been shown that these bands are derived from one gene by differential splicing (Gulcher et al., 1989). The three bands observed for undulin isolated from skin and placenta all reacted similarly when probed with one of several individual monoclonal antibodies in immunoblots. Therefore it appears that they are derived from a common gene either by differential splicing of a parent mRNA or by extensive posttranslational modifications of a single nascent polypeptide chain. Since only one mRNA of approximately 7 kb is found in Northern blots of preparations from both human placenta and a rhabdomyosarcoma cell line using various different undulin cDNA clones," the latter possibility seems more plausible. The size of this mRNA is close to that of the human fibronectin message (two differentially spliced forms around 7.9 kb; Vibe-Pedersen et al., 1984), chicken tenascin/cytotactin (messages between 7 and 9 kb; Jones et al., 1988;Pearson et al., 1989)  of about 30% of human undulin from cyanogen bromide peptides and cDNA clones.' locally revealing up to 42% homology with regions in human fibronectin and up to 33% homology with parts of tenascin/cytotactin/hexabrachion, but showing no similarities with human thrombospondin or laminin, further underlining its unique nature as a distinct member of an ECM glycoprotein superfamily. Augmenting the common features of the subunit composition of tenascin/cytotactin/hexabrachion and undulin, a monoclonal antibody against mouse tenascin has recently been shown to react with all chains of M, 260,000, 210,000, and 200,000 of reduced tenascin in immunoblots of extracts of mouse embryonic and adult intestine (Aufderheide and Ekblom, 1988).
Despite the above-mentioned similarities between undulin and tenascin the data suggest divergent biological roles for the two molecules. In all tissues studied so far their distributions have been found to be mutually exclusive. Tenascin/ cytotactin/hexabrachion is expressed in mesenchymal tissues interacting with differentiating or rapidly growing epithelia such as during limb bud morphogenesis (Chiquet and Farmbrough, 1984), kidney tubule formation (Aufderheide et al., 1987), early brain cell migration (Crossin et al., 1986), epithelial cell renewal in the gut (Aufderheide and Ekblom, 1988), and cancerous infiltration by mammary tumors (Mackie et al., 1987). Undulin, in contrast, is virtually absent underneath by guest on March 24, 2020 http://www.jbc.org/ Downloaded from epithelia subject to rapid cell renewal and is rapidly degraded in the vicinity of invading tumors such as Kaposi sarcoma and oral squamous cell carcinomas.3 Since undulin is primarily associated with a particular subgroup of collagen fibrils some speculation about its biological function mav be made. The fibrils with which undulin is associated appear highly ordered in uniform bundles. These bundles are prominent in tissues like skin and oral mucosa and at the periphery of structures like blood vessels, glands, and glandular ducts where high mechanical flexibility combined with stabilitv are imnortant.
In keening with the ultra-I Y structural data which show undulin preferentially in regions of densely packed collagen fibers, it may be hypothesized that the glycoprotein acts as a flexible anchor allowing limited gliding between individual collagen fibrils. The lack of undulin in bone and its paucity in scar tissue (not shown) are in good agreement with this hypothesis. Furthermore, undulin antibodies label only highly uniform fibers representing bundles of collagen fibrils, thereby yielding a regularity of pattern not seen for any other ECM component.
Thus it may be speculated that undulin is somehow involved in the suuramolecular organization of collagen fibrils characteristic of differentiated mesenchyme, but lost or reduced, for example, when irreversible fibrosis of the liver and when tumorous invasion of the interstitial ECM occur. In this regard Birk and Trelstad (1984) postulated that coalescence of mature collagen fibrils in fibroblast surface folds necessitates the incorporation of another component, possibly a glycoprotein.
Whether undulin fulfills such a hypothetical function as well as the nature of the interaction of undulin and collagen fibrils remain to be clarified in the future. The anomalous binding of type I collagen to DEAE-cellulose in the presence of undulin, their coelution during purification, and our recent finding of a specific interaction of mainly the a2 chain of type I collagen with undulin" attest to the innate affinity of the two molecules.
Undulin is produced by a variety of cell types in uitro. Low levels are found in cultures of human skin fibroblasts and a high rate of synthesis is found in a mesenchymal tumor cell line, whereas no undulin has been detected in epithelial cell cultures. In these cultures and in extracts from a variety of human tissues a remarkably constant electrophoretic pattern has been observed with band A (Mr 270,000) and a broad band B (Mr 180,000) after reduction, which indicates a fairly uniform processing of the presumed parent protein and/or mRNA.
The association of undulin with the surface of mature collagen fibrils and the finding that it appears to be removed more rapidly than any other of the major ECM components during carcinomatous invasion could qualify undulin or its fragments as key molecules for biochemical, histological, and serological studies on tissue differentiation and metastasis.