Advertisement

EMILIN, a Component of the Elastic Fiber and a New Member of the C1q/Tumor Necrosis Factor Superfamily of Proteins*

  • Roberto Doliana
    Affiliations
    Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico di Aviano, 33081 Aviano, Italy,
    Search for articles by this author
  • Maurizio Mongiat
    Affiliations
    Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico di Aviano, 33081 Aviano, Italy,
    Search for articles by this author
  • Francesco Bucciotti
    Affiliations
    Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico di Aviano, 33081 Aviano, Italy,
    Search for articles by this author
  • Emiliana Giacomello
    Affiliations
    Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico di Aviano, 33081 Aviano, Italy,
    Search for articles by this author
  • Rainer Deutzmann
    Affiliations
    Department of Biochemistry, Genetics and Microbiology, University of Regensburg, Regensburg, Germany D-8400,
    Search for articles by this author
  • Dino Volpin
    Affiliations
    Istituto di Istologia, Università di Padova, 35100 Padova, Italy, and
    Search for articles by this author
  • Giorgio M. Bressan
    Affiliations
    Istituto di Istologia, Università di Padova, 35100 Padova, Italy, and
    Search for articles by this author
  • Alfonso Colombatti
    Correspondence
    To whom correspondence should be addressed: Divisione di Oncologia Sperimentale, Centro di Riferimento Oncologico, 33081 Aviano, Italy. Fax: 0039 0434 659 428;
    Affiliations
    Divisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico di Aviano, 33081 Aviano, Italy,

    Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, 33100 Udine, Italy
    Search for articles by this author
  • Author Footnotes
    * This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, Telethon, MURST-Cofin 1997 (to A. C.), and Ricerca Sanitaria Finalizzata 793/03/97 (D.V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF088916.
Open AccessPublished:June 11, 1999DOI:https://doi.org/10.1074/jbc.274.24.16773
      EMILIN (elastinmicrofibril interface located protein) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as blood vessels, skin, heart, and lung. It occurs associated with elastic fibers at the interface between amorphous elastin and microfibrils. Avian EMILIN was extracted from 19-day-old embryonic chick aortas and associated blood vessels and purified by ion-exchange chromatography and gel filtration. Tryptic peptides were generated from EMILIN and sequenced, and degenerate inosine-containing oligonucleotide primers were designed from some peptides. A set of primers allowed the amplification of a 360-base pair reverse transcription polymerase chain reaction product from chick aorta mRNA. A probe based on a human homologue selected by comparison of the chick sequence with EST data base was used to select overlapping clones from both human aorta and kidney cDNA libraries. Here we present the cDNA sequence of the entire coding region of human EMILIN encompassing an open reading frame of 1016 amino acid residues. There was a high degree of homology (76% identity and 88% similarity) between the chick C terminus and the human sequence as well as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the C terminus, a collagenous stalk, and a longer segment in which at least four heptad repeats and a leucine zipper can be identified with a high potential for forming coiled-coil α helices. At the N terminus there is a cysteine-rich sequence stretch similar to a region of multimerin, a platelet and endothelial cell component, containing a partial epidermal growth factor-like motif. The native state of the recombinantly expressed EMILIN C1q-like domain to be used in cell adhesion was determined by CD spectra analysis, which indicated a high value of β-sheet conformation. The EMILIN C1q-like domain promoted a high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the fibrosarcoma cell line HT1080 was negative.
      Elastic fibers are major constituents of the extracellular matrix (ECM),
      The abbreviations used are: ECM, extracellular matrix; gC1q-like, globular C1q-like domain; TNF, tumor necrosis factor; PCR, polymerase chain reaction; EST, Expressed Sequence Tag; bp, base pair(s); RACE, rapid amplification of cDNA ends; CAFCA, centrifugal assay for fluorescence-based cell adhesion
      1The abbreviations used are: ECM, extracellular matrix; gC1q-like, globular C1q-like domain; TNF, tumor necrosis factor; PCR, polymerase chain reaction; EST, Expressed Sequence Tag; bp, base pair(s); RACE, rapid amplification of cDNA ends; CAFCA, centrifugal assay for fluorescence-based cell adhesion
      confer to connective tissues the properties of resilience and elastic recoil, and are remarkable for the diverse range of tissues in which they are found. Elastic fibers can be identified in the ECM of many tissues as solid branching and unbranching fine and thick rod-like fibers (in elastic ligaments) or as concentric sheets of lamellae (in blood vessels) or in three-dimensional meshworks of fine fibrils (in elastic cartilage) or as a combination of these (in skin and lung) (
      • Partridge S.M.
      ). Electron microscopy has provided additional insights into the structure of elastic fibers, which are composed of two morphologically distinguishable components: an amorphous core lacking any apparent regular or repeating structure (
      • Greenlee Jr., T.K.
      • Ross R.
      • Hartman J.L.
      ) and a microfibrillar component (
      • Cleary E.G.
      ) consisting of fibrils of 12–13 nm in diameter that are located primarily around the periphery of the amorphous core but to some extent are also interspersed within it.
      Despite this simple morphology, elastic fibers are now known to be highly complex structures (
      • Rosenbloom J.
      • Abrams W.R.
      • Mecham R.
      ) consisting of several specific proteins, including elastin, fibrillin 1 and 2 (
      • Sakay L.Y.
      • Keene D.R.
      • Engvall E.
      ,
      • Zhang H.
      • Apfelroth S.D.
      • Hu W.
      • Davis E.E.
      • Sanguineti C.
      • Bonadio J.
      • Mecham R.P.
      • Ramirez F.
      ), microfibril-associated proteins 1 to 4 (
      • Henderson M.
      • Polewski R.
      • Fanning J.C.
      • Gibson M.A.
      ,
      • Gibson M.A.
      • Hatzinikolas G.
      • Kumaratilake J.S.
      • Sandberg L.B.
      • Nicholl J.K.
      • Sutherland G.R.
      • Cleary E.G.
      ,
      • Abrams W.R.
      • Ma R.I.
      • Kucich U.
      • Bashir M.M.
      • Decker S.
      • Tsipouras P.
      • McPherson J.D.
      • Wasmuth J.J.
      • Rosenbloom J.
      ,
      • Zhao Z.
      • Lee C.-C.
      • Jiralerspong S.
      • Juyal R.C.
      • Lu F.
      • Baldini A.
      • Greenberg F.
      • Caskey C.T.
      • Patel P.I.
      ), latent-transforming growth factor β-binding protein 1 to 4 (
      • Kanzaki T.
      • Olofsson A.
      • Moren A.
      • Werntedt C.
      • Hellman U.K.
      • Claesson-Welsh L.
      • Heldin C.H.
      ,
      • Gibson M.A.
      • Hatzinikolas G.
      • Davis E.C.
      • Baker E.
      • Sutherland G.R.
      • Mecham R.P.
      ,
      • Yin W.
      • Smiley E.
      • Germiller J.
      • Mecham R.P.
      • Florer J.R.
      • Wenstrup R.J.
      • Bonadio J.
      ,
      • Saharinen J.
      • Taipale J.
      • Monni O.
      • Keski-Oja J.
      ), fibulin (
      • Roak E.F.
      • Keene D.R.
      • Haudenschild C.C.
      • Godyna S.
      • Little C.D.
      • Argraves W.S.
      ), and MAGP-2 (
      • Gibson M.A.
      • Hatzinikolas G.
      • Kumaratilake J.S.
      • Sandberg L.B.
      • Nicholl J.K.
      • Sutherland G.R.
      • Cleary E.G.
      ,
      • Hatzinikolas G.
      • Gibson M.A.
      ). Elastin and fibrillins are the main components of the amorphous core and microfibrils, respectively. Fibrillin-containing microfibrils are highly organized structures, and microfibril-associated proteins and probably latent-transforming growth factor β-binding proteins are predominantly associated with them. Fibrillin-containing microfibrils are also found as elastin-free bundles in tissues where elastic fibers cannot be morphologically distinguished such as in ocular zonule, oxitalan fibers of the cornea, kidney, and spleen (
      • Cleary E.G.
      • Gibson M.A.
      ).
      We had originally isolated from chick aorta a novel glycoprotein component associated with the ECM of blood vessels, gp115 (
      • Bressan G.M.
      • Castellani I.
      • Colombatti A.
      • Volpin D.
      ), later christened EMILIN (
      • Bressan G.M.
      • Daga-Gordini D.
      • Colombatti A.
      • Castellani I.
      • Marigo V.
      • Volpin D.
      ). The major characteristics of this protein were the following. EMILIN was preferentially extracted from tissues using buffers containing guanidine HCl and reducing agents; it formed a fibrillar network in the ECM of aorta; the amino acid composition was characterized by a high content of glutamic acid and arginine (
      • Bressan G.M.
      • Castellani I.
      • Colombatti A.
      • Volpin D.
      ). Subsequent studies have established that (i) EMILIN is broadly expressed in connective tissues and is particularly abundant in blood vessels, skin, heart, lung, kidney, and cornea, whereas it is undetectable in the serum (
      • Colombatti A.
      • Bressan G.M.
      • Castellani I.
      • Volpin D.
      ,
      • Colombatti A.
      • Bressan G.M.
      • Volpin D.
      • Castellani I.
      ,
      • Colombatti A.
      • Poletti A.
      • Bressan G.M.
      • Carbone A.
      • Volpin D.
      ); (ii) the protein is synthesized by aortic smooth muscle cells and by tendon fibroblasts, and it is deposited extracellularly as a fine network (
      • Bressan G.M.
      • Castellani I.
      • Colombatti A.
      • Volpin D.
      ,
      • Colombatti A.
      • Bonaldo P.
      • Volpin D.
      • Bressan G.M.
      ); (iii) soon after secretion, EMILIN undergoes intermolecular cross-linking by disulfide bonds, giving rise to high molecular weight aggregates (
      • Colombatti A.
      • Bonaldo P.
      • Volpin D.
      • Bressan G.M.
      ); (iv) EMILIN is a component of elastic fibers and is localized mainly at the interface between amorphous elastin and microfibrils (
      • Bressan G.M.
      • Daga-Gordini D.
      • Colombatti A.
      • Castellani I.
      • Marigo V.
      • Volpin D.
      ); (v) finally and more important for the functional significance of EMILIN, the process of elastin deposition in vitro is perturbed by the addition of anti-EMILIN antibodies in the culture medium (
      • Bressan G.M.
      • Daga-Gordini D.
      • Colombatti A.
      • Castellani I.
      • Marigo V.
      • Volpin D.
      ). Therefore, given the close co-distribution of elastin and EMILIN, the fine localization at the interface between elastin and microfibrils, and the interference with the deposition of elastin in vitro, it is likely that EMILIN plays a fundamental role in the process of elastogenesis also in vivo. To initiate addressing the question of the functional role of EMILIN directly, we have sought to clone its gene.
      We report here the cDNA sequence and the analysis of the deduced amino acid sequence of human EMILIN. This glycoprotein, a new member of the C1q/TNF superfamily of proteins, is characterized by a gC1q-like C-terminal domain, a short collagenous domain, two leucine zippers, and an extended coiled-coil region that is uniquely shared with another member of this superfamily, multimerin (
      • Hayward C.P.M.
      • Hassell J.A.
      • Denomme G.A.
      • Rachubinski R.A.
      • Brown C.
      • Kelton J.G.
      ). At the N terminus of these two members of the superfamily there is a short region of homology including a partial epidermal growth factor-like motif. In addition, the isolated recombinantly produced EMILIN gC1q-like C-terminal domain is able to support cell adhesion.

      DISCUSSION

      The results provided in this report concern a new human cDNA whose major structural elements were a gC1q-like C-terminal domain, a short uninterrupted collagenous domain, and an extended domain containing sequences with the potential of forming amphipathic coiled-coil α-helices. The determination of the primary structure through cDNA cloning and the assignment of this novel sequence to human EMILIN was made possible by the very close identity at the C terminus between the chick EMILIN sequence and that of the corresponding human cDNA. In fact, the finding that the deduced sequence of clone D1 of chick EMILIN contained peptide 1 of the tissue-purified EMILIN confirmed that the sequence amplified from chick aorta mRNA corresponded to that of the genuine EMILIN described by us (
      • Bressan G.M.
      • Castellani I.
      • Colombatti A.
      • Volpin D.
      ,
      • Bressan G.M.
      • Daga-Gordini D.
      • Colombatti A.
      • Castellani I.
      • Marigo V.
      • Volpin D.
      ,
      • Colombatti A.
      • Bressan G.M.
      • Castellani I.
      • Volpin D.
      ,
      • Colombatti A.
      • Bressan G.M.
      • Volpin D.
      • Castellani I.
      ,
      • Colombatti A.
      • Poletti A.
      • Bressan G.M.
      • Carbone A.
      • Volpin D.
      ,
      • Colombatti A.
      • Bonaldo P.
      • Volpin D.
      • Bressan G.M.
      ) as an elastin-associated protein with undefined function. Thus, the sequence similarity at the gC1q-like domain, the near identity between the N-terminal residues of the mature chick protein, and the deduced amino acids of the presumed mature human protein support the conclusion that we are dealing with the same ECM constituent in the two species.
      Evidence that chick EMILIN stained strongly with PAS (
      • Bressan G.M.
      • Castellani I.
      • Colombatti A.
      • Volpin D.
      ) and that in biosynthetic studies a treatment with tunicamycin reduced the apparent molecular mass of about 20–25 kDa (
      • Colombatti A.
      • Bonaldo P.
      • Volpin D.
      • Bressan G.M.
      ) indicated that chick EMILIN was highly glycosylated. The present identification of seven potentialN-glycosylation sites in the human EMILIN sequence is in accord with the previous experimental data using the chick system (
      • Bressan G.M.
      • Castellani I.
      • Colombatti A.
      • Volpin D.
      ,
      • Colombatti A.
      • Bonaldo P.
      • Volpin D.
      • Bressan G.M.
      ). Similarly, the presence of 20 cysteine residues with a high potential for intermolecular S-S bonding is also in accord with the finding that newly synthesized and secreted chick EMILIN migrated as a monomer in SDS gels under reduced conditions but was present as a large aggregate that did not enter the gel in the absence of reducing agents (
      • Colombatti A.
      • Bonaldo P.
      • Volpin D.
      • Bressan G.M.
      ).
      The domain organization of EMILIN is unique; it bears features shared with several other members of the C1q/TNF superfamily (Fig.9), i.e. C1q (A, B, C), collagens VIII, X, saccular collagen, ACRP-30/AdipoQ, and HP-27, such as the gC1q-like domain and a collagenous domain, but also EMILIN displays an extended discontinuous and potentially coiled-coil region that is absent in all the other members of the C1q/TNF superfamily, except multimerin, a large soluble glycoprotein found in platelets α-granules and endothelial Weibel-Palade bodies. Multimerin forms disulfide-linked homomultimers of variable sizes (
      • Hayward C.P.M.
      • Warkentin T.E.
      • Horsewood P.
      • Kelton J.G.
      ) and interacts with factor V, which is stored complexed with multimerin in the α-granules (
      • Hayward C.P.M.
      • Furmaniak-Kazmierczak E.
      • Cieutat A.-M.
      • Moore J.C.
      • Bainton D.F.
      • Nesheim M.E.
      • Kelton J.G.
      • Coté G.
      ). There is experimental evidence that several members of the C1q/TNF superfamily trimerize to form either heterotrimeric collagen triple helices that are expressed as soluble plasma proteins or type II membrane-bound molecules such as in C1q (A, B, C) (
      • Reid K.B.
      ,
      • Kaul M.
      • Loos M.
      ) or to form homotrimers as in collagen X (
      • Frischoltz S.
      • Beier F.
      • Girkontaite I.
      • Wagner K.
      • Poschl E.
      • Turnay J.
      • Mayer U.
      • von der Mark K.
      ) and ACRP-30/AdipoQ (
      • Hu E.
      • Liang P.
      • Spiegelman B.M.
      ). EMILIN is also likely to form similar trimers; it possesses a gC1q-like domain, which is highly homologous to those of the other members of the superfamily and an uninterrupted collagenous domain, which can form a collagen-like stalk region. Curiously, among the members of the family, only EMILIN, ACRP-30/AdipoQ, and Hib27 possess an uninterrupted collagenous domain. The EMILIN gC1q-like domain, when compared with the other gC1q-like domains, has a much longer F β-strand because of a 10-residue insertion. However, the residues conserved throughout both the C1q and TNF families of proteins and important in the packing of the hydrophobic core of the individual monomer (
      • Shapiro L.
      • Scherer P.E.
      ) are present in EMILIN gC1q-like domain in the same relative positions, and this appears sufficient to predict a similar trimeric and spatial organization also for the domain of EMILIN. The potential structural homology between the TNF family of growth factors and the gC1q-like domains of the C1q family of proteins suggested that these diverse members might derive from ancestral elements with close functional activity (
      • Shapiro L.
      • Scherer P.E.
      ). Likely targets for the proteins containing C1q/TNF domains are cell surface receptors; these are well studied in TNF, but initial data are also available for the C1q complement component (
      • Ghebrehiwet B.
      • Boon-Leong L.
      • Peerschke E.I.
      • Willis A.C.
      • Reid K.B.M.
      ,
      • Peerschke E.I.
      • Reid K.B.M.
      • Ghebrehiwet B.
      ,
      • Peerschke E.I.
      • Smyth S.S.
      • Teng E.I.
      • Dalzell M.
      • Ghebrehiwet B.
      ). In fact, several cell types are endowed with the capability to attach to the C1q complement component via cell surface binding sites; two types of structures have been described, a binding protein that recognizes the collagenous domain (
      • Peerschke E.I.
      • Malhotra R.
      • Ghebrehiwet B.
      • Reid K.B.M.
      • Willis A.C.
      • Sim R.B.
      ) and another component that binds to the gC1q domain (
      • Ghebrehiwet B.
      • Boon-Leong L.
      • Peerschke E.I.
      • Willis A.C.
      • Reid K.B.M.
      ). However, more recently the effective nature of this second type of binding molecule has been disputed (
      • Dedio J.
      • Jahnen-Dechent W.
      • Bachmann M.
      • Muller-Esterl W.
      ), and further studies are necessary. The finding that EMILIN gC1q-like domain displayed cell pro-adhesive capacity for some smooth muscle cells but seemed to be much less reactive for fibroblastic cells is consistent with the above evidence and suggests that cell recognition of this domain might be exerted via specific cell surface receptors. The adhesion was high for SK-UT-1 cells but, within the time frame of the cell adhesion assay, was not followed by a consistent spreading. Thus, it is possible that “receptors” distinct from classical integrins such as those recognized by fibronectin are involved here. Neither the physiological significance of the observed adhesion is clear yet nor whether this adhesion plays a primary or an auxiliary role. Close contacts between amorphous elastin and smooth muscle cells in the aorta of 16-day embryos have been reported (
      • Daga-Gordini D.
      • Bressan G.M.
      • Castellani I.
      • Volpin D.
      ), and the ultrastructural localization of EMILIN (
      • Bressan G.M.
      • Daga-Gordini D.
      • Colombatti A.
      • Castellani I.
      • Marigo V.
      • Volpin D.
      ) does not exclude that the interaction between the elastin amorphous core and the cells could also take place via an EMILIN intermediate. As EMILIN was detected in early stages of aorta development, in association with a network of thin fibrils likely representing maturing microfibrils (
      • Bressan G.M.
      • Daga-Gordini D.
      • Colombatti A.
      • Castellani I.
      • Marigo V.
      • Volpin D.
      ), EMILIN deposition can be considered an early event in elastogenesis, and this conclusion is reinforced by the observation that the process of elastic fiber formation in vitro was greatly affected by the addition of anti EMILIN antibodies (
      • Bressan G.M.
      • Daga-Gordini D.
      • Colombatti A.
      • Castellani I.
      • Marigo V.
      • Volpin D.
      ). Whether the process of elastin deposition and elastic fiber formation is regulated through cell adhesion via the EMILIN gC1q-like domain remains to be seen.
      Figure thumbnail gr9
      Figure 9The C1q/TNF superfamily of proteins. The different domains are designated as in Fig. . Bars within the collagenous domains (COL) indicate short interruptions or imperfections in the Gly-Xaa-Yaa sequence. The order in which the different members are depicted highlights that EMILIN, in addition to the C1q-like domain common to all the family members, shears a short collagenous domain with only a few of the members and the coiled-coil-containing region only with multimerin. EGF, epidermal growth factor.
      EMILIN, like multimerin (
      • Hayward C.P.M.
      • Warkentin T.E.
      • Horsewood P.
      • Kelton J.G.
      ), is heavily disulfide-linked and thus can be found as large aggregates in the culture medium of aorta smooth muscle cells (
      • Colombatti A.
      • Bonaldo P.
      • Volpin D.
      • Bressan G.M.
      ). The possibility to form coiled-coil α-helices could further amplify its potential to associate into even larger aggregates. In fact, one of the heptad repeat sequences has a probability to form α-helices near 1.0, and in two other regions the probability is above 0.6. Although formal proof that these heptad repeats can form trimers is still lacking, the chances are high given that the presumed trimerization process can initiate from the C1q-like domain at the C terminus, proceeding then through the collagenous domain next to it as in collagen X (
      • Frischoltz S.
      • Beier F.
      • Girkontaite I.
      • Wagner K.
      • Poschl E.
      • Turnay J.
      • Mayer U.
      • von der Mark K.
      ) and ACRP-30/AdipoQ (
      • Hu E.
      • Liang P.
      • Spiegelman B.M.
      ). Further studies are required to define how EMILIN subunits are assembled into the large disulfide-linked multimers, i.e.whether the EMILIN gC1q-like domain is a likely site for initial interchain association and whether the heptad repeats associate only intramolecularly into trimers or can also associate intermolecularly,i.e. with other EMILIN trimers. To investigate these possibilities, the preparation of full-length, truncated, and point-mutated EMILIN recombinant molecules in eukaryotic cells is in progress.
      M. Mongiat, R. Doliana, and A. Colombatti, unpublished observations.

      ACKNOWLEDGEMENTS

      We thank Maria Teresa Mucignat and Gabriella Mungiguerra for technical assistance, Dr. Paola Spessotto for her help in performing the cell adhesion assays, and Dr. Gianluca Tell for performing the CD spectra analysis.

      REFERENCES

        • Partridge S.M.
        Adv. Protein Chem. 1962; 17: 227-302
        • Greenlee Jr., T.K.
        • Ross R.
        • Hartman J.L.
        J. Cell Biol. 1966; 30: 59-71
        • Cleary E.G.
        Uitto J. Parejda A.J. Connective Tissue Disease. Marcel Dekker, Inc., New York1987: 55-81
        • Rosenbloom J.
        • Abrams W.R.
        • Mecham R.
        FASEB J. 1993; 7: 1208-1218
        • Sakay L.Y.
        • Keene D.R.
        • Engvall E.
        J. Cell Biol. 1986; 103: 2499-2509
        • Zhang H.
        • Apfelroth S.D.
        • Hu W.
        • Davis E.E.
        • Sanguineti C.
        • Bonadio J.
        • Mecham R.P.
        • Ramirez F.
        J. Cell Biol. 1994; 124: 855-863
        • Henderson M.
        • Polewski R.
        • Fanning J.C.
        • Gibson M.A.
        J. Histochem. Cytochem. 1996; 44: 1389-1397
        • Gibson M.A.
        • Hatzinikolas G.
        • Kumaratilake J.S.
        • Sandberg L.B.
        • Nicholl J.K.
        • Sutherland G.R.
        • Cleary E.G.
        J. Biol. Chem. 1996; 271: 1096-1103
        • Abrams W.R.
        • Ma R.I.
        • Kucich U.
        • Bashir M.M.
        • Decker S.
        • Tsipouras P.
        • McPherson J.D.
        • Wasmuth J.J.
        • Rosenbloom J.
        Genomics. 1995; 26: 47-54
        • Zhao Z.
        • Lee C.-C.
        • Jiralerspong S.
        • Juyal R.C.
        • Lu F.
        • Baldini A.
        • Greenberg F.
        • Caskey C.T.
        • Patel P.I.
        Hum. Mol. Gen. 1995; 4: 589-597
        • Kanzaki T.
        • Olofsson A.
        • Moren A.
        • Werntedt C.
        • Hellman U.K.
        • Claesson-Welsh L.
        • Heldin C.H.
        Cell. 1990; 6: 1051-1061
        • Gibson M.A.
        • Hatzinikolas G.
        • Davis E.C.
        • Baker E.
        • Sutherland G.R.
        • Mecham R.P.
        Mol. Cell. Biol. 1995; 15: 6932-6942
        • Yin W.
        • Smiley E.
        • Germiller J.
        • Mecham R.P.
        • Florer J.R.
        • Wenstrup R.J.
        • Bonadio J.
        J. Biol. Chem. 1995; 270: 10147-10160
        • Saharinen J.
        • Taipale J.
        • Monni O.
        • Keski-Oja J.
        J. Biol. Chem. 1998; 273: 18459-18469
        • Roak E.F.
        • Keene D.R.
        • Haudenschild C.C.
        • Godyna S.
        • Little C.D.
        • Argraves W.S.
        J. Histochem. Cytochem. 1995; 43: 401-411
        • Hatzinikolas G.
        • Gibson M.A.
        J. Biol. Chem. 1998; 273: 29309-29314
        • Cleary E.G.
        • Gibson M.A.
        Comper W.D. The Structure and Function of Extracellular Matrix. 2. Gordon and Breach Science Publisher, New York1996: 95-140
        • Bressan G.M.
        • Castellani I.
        • Colombatti A.
        • Volpin D.
        J. Biol. Chem. 1983; 258: 13262-13267
        • Bressan G.M.
        • Daga-Gordini D.
        • Colombatti A.
        • Castellani I.
        • Marigo V.
        • Volpin D.
        J. Cell Biol. 1993; 121: 201-212
        • Colombatti A.
        • Bressan G.M.
        • Castellani I.
        • Volpin D.
        J. Cell Biol. 1985; 100: 18-26
        • Colombatti A.
        • Bressan G.M.
        • Volpin D.
        • Castellani I.
        Collagen Relat. Res. 1985; 5: 181-191
        • Colombatti A.
        • Poletti A.
        • Bressan G.M.
        • Carbone A.
        • Volpin D.
        Collagen Relat. Res. 1987; 7: 259-275
        • Colombatti A.
        • Bonaldo P.
        • Volpin D.
        • Bressan G.M.
        J. Biol. Chem. 1988; 263: 17534-17540
        • Hayward C.P.M.
        • Hassell J.A.
        • Denomme G.A.
        • Rachubinski R.A.
        • Brown C.
        • Kelton J.G.
        J. Biol. Chem. 1995; 270: 18246-18251
        • Pearson W.R.
        • Lipman D.J.
        Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448
        • Altschul S.F.
        • Gish W.
        • Miller M.
        • Myers E.W.
        • Lipman D.J.
        J. Mol. Biol. 1990; 215: 403-410
        • Menendez-Arias L.
        • Gomez-Gutierrez J.
        • Garcia-Fernandez M.
        • GarciaTejedor A.
        • Moran F.
        Comput. Appl. Biosci. 1989; 4: 479-482
        • Giacomello E.
        • Neumayer J.
        • Colombatti A.
        • Perris R.
        Biotechniques. 1999; (in press)
        • Spessotto P.
        • Giacomello E.
        • Perris R.
        Methods in Molecular Biology: Extracellular Matrix. Humana Press Inc., Totowa, NJ1999 (in press)
        • Kozak M.
        Nucleic Acids Res. 1981; 9: 5233-5252
        • Nielsen H.
        • Engelbrecht J.
        • Brunak S.
        • van Heijne G.
        Protein Eng. 1997; 10: 1-6
        • Perlman D.
        • Halvorson H.O.
        J. Mol. Biol. 1983; 167: 391-409
        • Landshultz W.H.
        • Johnson P.F.
        • McKnight S.L.
        Science. 1988; 240: 1759-1764
        • Bush S.J.
        • Sassone-Corsi P.
        Trends Genet. 1990; 6: 36-40
        • Zhang S.-D.
        • Kassis J.
        • Olde B.
        • Mellerick D.M.
        • Odenwald W.F.
        Genes Dev. 1996; 10: 1108-1119
        • Pearlman J.A.
        • Powaser P.A.
        • Elledge S.J.
        • Caskey C.T.
        FEBS Lett. 1994; 354: 183-186
        • Lupas A.
        Methods Enzymol. 1996; 266: 513-525
        • Berger B.
        • Wilson D.B.
        • Wolf E.
        • Tonchev T.
        • Milla M.
        • Kim P.S.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8259-8263
        • Sellar G.C.
        • Blake D.J.
        • Reid K.B.M.
        Biochem. J. 1991; 274: 481-490
        • Reid K.B.M.
        Biochem. J. 1985; 231: 729-735
        • Muragaki Y.
        • Mattel M.-G.
        • Yamaguchi N.
        • Olsen B.R.
        • Ninomiya Y.
        Eur. J. Biochem. 1991; 197: 615-622
        • Muragaki Y.
        • Jacenko O.
        • Apte S.
        • Mattel M.-G.
        • Ninomiya Y.
        • Olsen B.R.
        J. Biol. Chem. 1991; 266: 7721-7727
        • Reichenberger E.
        • Beler F.
        • LuValle P.
        • Olsen B.R.
        • von der Mark K.
        • Bertling W.M.
        FEBS Lett. 1992; 311: 305-310
        • Urade Y.
        • Oberdick J.
        • Molinar-Rode R.
        • Morgan J.I.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1069-1073
        • Scherer P.E.
        • Williams S.
        • Fogliano M.
        • Baldini G.
        • Lodish H.F.
        J. Biol. Chem. 1995; 270: 26746-26749
        • Hu E.
        • Liang P.
        • Spiegelman B.M.
        J. Biol. Chem. 1996; 271: 10697-10703
        • Takamatsu N.
        • Ohba K.
        • Kondo J.
        • Kondo N.
        • Shiba T.
        Mol. Cell. Biol. 1993; 13: 1516-1521
        • Davis J.G.
        • Burns F.R.
        • Navaratnam D.
        • Lee A.M.
        • Ichimiya S.
        • Oberholtzer J.C.
        • Greene M.I.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 707-712
        • Smith K.F.
        • Haris P.I.
        • Chapman D.
        • Reid K.B.M.
        • Perkins S.J.
        Biochem. J. 1994; 301: 249-256
        • Shapiro L.
        • Scherer P.E.
        Curr. Biol. 1998; 8: 335-338
        • Ghebrehiwet B.
        • Boon-Leong L.
        • Peerschke E.I.
        • Willis A.C.
        • Reid K.B.M.
        J. Exp. Med. 1994; 179: 1809-1821
        • Peerschke E.I.
        • Reid K.B.M.
        • Ghebrehiwet B.
        J. Immunol. 1994; 152: 5896-5901
        • Peerschke E.I.
        • Smyth S.S.
        • Teng E.I.
        • Dalzell M.
        • Ghebrehiwet B.
        J. Immunol. 1996; 157: 4154-4158
        • Bork P.
        • Koonin E.V.
        Curr. Opin. Struct. Biol. 1996; 6: 366-375
        • Hayward C.P.M.
        • Warkentin T.E.
        • Horsewood P.
        • Kelton J.G.
        Blood. 1991; 77: 2556-2560
        • Hayward C.P.M.
        • Furmaniak-Kazmierczak E.
        • Cieutat A.-M.
        • Moore J.C.
        • Bainton D.F.
        • Nesheim M.E.
        • Kelton J.G.
        • Coté G.
        J. Biol. Chem. 1995; 270: 19217-19224
        • Reid K.B.
        Behring Inst. Mitt. 1989; 84: 8-19
        • Kaul M.
        • Loos M.
        J. Immunol. 1995; 155: 5795-5802
        • Frischoltz S.
        • Beier F.
        • Girkontaite I.
        • Wagner K.
        • Poschl E.
        • Turnay J.
        • Mayer U.
        • von der Mark K.
        J. Biol. Chem. 1998; 273: 4547-4555
        • Peerschke E.I.
        • Malhotra R.
        • Ghebrehiwet B.
        • Reid K.B.M.
        • Willis A.C.
        • Sim R.B.
        J. Leukocyte Biol. 1993; 53: 179-184
        • Dedio J.
        • Jahnen-Dechent W.
        • Bachmann M.
        • Muller-Esterl W.
        J. Immunol. 1998; 160: 3534-3542
        • Daga-Gordini D.
        • Bressan G.M.
        • Castellani I.
        • Volpin D.
        Histochem. J. 1987; 19: 623-632