Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding.

Abstract

Abstract.A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a l195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a ll5-residue globular domain HI. Except for two additional EGF-like repeats, this COOH-terminal region showed 43 % sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.

New insights into elastic fiber assembly

“…Fibulin-1 associates with some basement membranes and with the amorphous elastin core of elastic fibers, but not with individual microfibrils (Roark et al, 1995). Fibulin-2 also associates with basement membranes (Pan et al, 1993) and strongly binds tropoelastin (Sasaki et al, 1999). Unlike fibulin-1, fibulin-2 binds directly to fibrillin-1 and has been shown to localize to the interface between the amorphous elastin core and microfibrils in skin .…”

Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma

“…Polyclonal antiserum against the full-length FBLN2 protein was prepared as described (Pan et al, 1993). The a-tubulin antibody Ab-1 (1:10 000; Merck, Darmstadt, Germany) was used as a normalization control.…”

Fibulin-2 Is Dispensable for Mouse Development and Elastic Fiber Formation

“…Immunohistochemistry was performed using 6-to 8-m-thick cryosections of mouse tissues or fibroblasts grown on chamber slides by the method described elsewhere (26). Primary antibodies included those specific for each of the five fibulins (1:1,000 dilution) (8,17), fibrillin-1 (a generous gift of Lynn Sakai), elastin (PR387; Elastin Products Company, Owensville, MO), and fibronectin (Sigma, St. Louis, MO). Cy3-conjugated anti-rabbit immunoglobulin G (1: 800 dilution; Jackson ImmunoResearch Laboratory, West Grove, PA) was used as the secondary antibody.…”

“…Fibulin-1 and fibulin-2 contain an additional domain of three anaphylatoxin modules preceding the calcium-binding epidermal growth factor-like repeats and therefore are larger than fibulin-3, -4, and -5, which have identical modular structures. Fibulin-2 possesses a large aminoterminal globular domain of approximately 400 amino acids that is not present in the other fibulins and hence is the largest member of the family (17). The protein is approximately 180 kDa in size and forms covalently linked dimers (22).…”