Abstract. We have studied the expression of fibulin in cultured fibroblasts and determined its primary structure by eDNA cloning. Our results show that fibulin is a secreted glycoprotein that becomes incorporated into a fibriUar extracellular matrix when expressed by cultured cells or added exogenously to cell monolayers. In addition, we find that fibulin is present in plasma at a level of 33 + 3/~g/ml. Sequencing of multiple fibulin cDNAs indicates that a process of alternative splicing results in the expression of three fibulin transcripts. The transcripts encode overlapping polypeptides differing only in carboxy-terminal segments. Common to the three predicted forms of fibulin is a unique 537-amino acid-long cysteine-rich polypeptide and a 29-residue signal peptide. The amino-terminal portion of fibulin contains a repeated element with potential disulfide loop structure resembling that of the complement component anaphylatoxins C3a, C4a, and C5a as well as proteins of the albumin gene family. The bulk of the remaining portion of the molecule is a series of nine EGF-like repeats.F mULIN is a recently described calcium-binding protein which has been shown to interact with a synthetic peptide representing the cytoplasmic domain of the integrin/~1 subunit as well as native ot5/3~ fibronectin receptor (Argraves et al., 1989). Indirect immunofluorescent staining of cultured fibroblasts revealed that fibulin colocalized with the integrin/3~ subunit, in vivo, at sites of cellular interaction with underlying fibronectin substratum. It was therefore suspected that fibulin might be an intracellular protein involved with mediating cytoplasmic connections of the /31 integrins. Herein we report the results of characterization of fibulin expression and structure. The results indicate that fibulin is glycosylated and secreted by cultured fibroblasts and becomes incorporated into an extracellular matrix in a fashion similar to fibronectin. Salient structural features deduced from eDNA clones reveal that fibulin is a multidomain protein with two types of repeat motifs, one of which is homologous to the anaphylatoxins C3a, C4a, and C5a, as well as elements of proteins of the albumin gene family, and the other which is homologous to EGF. Materials and Methods AntibodiesThe following antisera were used for the immunoprecipitation and immunofluorescent staining experiments described herein: mouse monoclonal anti-human fibronectin (not cross-reactive with the bovine fibroneetin used in slide coating) was purchased from Telios Pharmaceuticals, La Jolla, CA; Kenneth Dickerson's present address is La Jolla Cancer Research Foundation, La JoUa, CA 92037. mouse monoelonal anti-human integrin #1 subunit was provided by Dr. E. Ruoslahti, La Jolla Cancer Research Foundation, La Jolla, CA; and rabbit anti-human fibulin serum was prepared in this laboratory and has been described previously (Argraves et al., 1989). As a precaution, the antifibulin serum used in the immunofluorescent staining experiments was absorbed on columns of human flbron...
Abstract. Ligand affinity chromatography was used to purify a cell surface a2-macroglobulin (ot2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of ot2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420-and 39-kD polypeptides appear specific for the forms of ~2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native ot2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of ot2M that are known to specifically interact with o~2M receptors and does not bind to native c~2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of ~zM.Ot2-MACROGLOBULIN (ot2M)' is a 718,000-mol wt glycoprotein that is capable of inhibiting enzymes from all four classes of proteinases (2) in a reaction that is essentially irreversible. The reaction of proteinases or methylamine with c~2M "activates" the molecule by inducing conformational changes in o~2M (3,4,16,49) that, in the case of the proteinase reaction, reduce the activity of the bound enzyme toward large molecular weight substrates. These conformational changes also generate sites on the inhibitor that result in specific recognition by cell surface receptors (31,52,54). It has been possible to prepare monoclonal antibodies that selectively recognize the activated forms of u2M (34, 50) by binding to these newly generated regions (25).In addition to regulating proteinase activity, studies have suggested that c~2M binds to TGFfl (41), PDGF (22), and bFGF (7). The association of these molecules with ot2M results in a reduction of their activity, suggesting that a2M may serve not only to modulate proteinase activity but also the activity of these molecules. Interestingly, it appears that several tumor-derived or virus-transformed cell lines are devoid of specific cell surface receptors for t~2M-proteinase complexes (52).The process of receptor-mediated endocytosis has been the subject of extensive study, and the mechanisms involved in I. Abbreviations used in this paper: ct2M, ~2-macroglobulin; t~2M:Me, methylamine-treated ~2-macroglobulin; NRK, normal rat kidney. endocytosis have been investigated using a variety of labeled ligands (6), including transferrin (20), low density lipoprotein (1), EGF (20), and ot2M (8,37,54,55). Early studies reported the specific bindi...
Considerable research effort has been directed at preparing root surfaces in a fashion that would promote cell attachment leading to periodontal regeneration; however, no methods have proven to be clinically predictable. Identification of attachment protein(s) associated with the root surface matrix of cementum may prove valuable for developing effective clinical treatments. In this study cementum proteins were extracted from bovine and human teeth by sequential chaotropic extraction using guanidine followed by guanidine/EDTA. The guanidine/EDTA extract, but not guanidine extract, was found to promote attachment of fibroblasts. This attachment activity was inhibitable with synthetic peptide containing the attachment sequence arginine-glycine-aspartic acid (RGD). Fractionation of the guanidine/EDTA extract revealed several fractions with attachment activity. Immunoblot analysis demonstrated that two of these fractions contain the bone-associated RGD containing attachment protein, bone sialoprotein-II (BSP-II). In addition, attachment activity was also noted in other fractions that could not be attributed to BSP-II or fibronectin. These studies indicate that a component of the attachment activity of cementum is likely to be due to BSP-II and that cementum contains additional, as yet undetermined, attachment proteins.
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