Biomedical subjects

W S Argraves

Publications and source records attributed to W S Argraves.

At least 19 recordsLinked to original sources

PubMed2003

Monoclonal antibody to fibulin-1 generated by genetic immunization.

Fibulin-1 (Fbln-1) is an extracellular matrix (ECM) and plasma glycoprotein. Considering the growing evidence indicating that Fbln-1 plays a role in cancer we sought to develop monospecific antibodies to better facilitate further studies of the function of Fbln-1 in breast cancer. Using a plasmid expression vector encoding full-length human Fbln-1D as an immunogen and CpG oligodeoxyribonucleotides as adjuvant a monoclonal antibody (MAb) against Fbln-1 was produced. This MAb, designated MEM-2 was of IgM isotype and reacted with bacterially expressed Fbln-1. Furthermore, MEM-2 reacted with Fbln-1 expressed in the ECM released by cultured human breast carcinoma SKBR-3 cells in ELISA, and also with Fbln-1 present in SKBR-3 cell extract in immunoprecipitation and Western blotting. MEM-2 also reacted with Fbln-1 in human breast carcinoma specimens. These findings illustrate the utility of genetic immunization as a means of generating monoclonal antibodies to tumor-related ECM proteins. MEM-2 represents a useful new tool for the study of Fbln-1 in breast cancer.

Adjuvants, Immunologic↗

PubMed2003

Elevated expression and altered processing of fibulin-1 protein in human breast cancer.

The extracellular matrix protein fibulin-1 suppresses the motility and invasiveness of a variety of tumour cell types in vitro as well as the growth of fibrosarcoma tumours in nude mice. In this study, fibulin-1 protein expression in breast carcinoma specimens and normal breast tissue was evaluated immunohistologically. Fibulin-1 protein expression was also semiquantitatively assessed by immunoblot analysis in a collection of normal breast tissues (n=18), benign tumours (n=5) and breast carcinomas (n=39). In normal breast tissue, fibulin-1 protein expression predominated in the ductal epithelium and underlying myoepithelium, with weaker staining evident in the loose connective surrounding the ducts. Examination of breast carcinomas revealed that the tumour cells also expressed fibulin-1 protein. The level of mature fibulin-1 polypeptide (100 kDa) was higher in the breast carcinoma specimens as compared to normal breast tissue (Mann-Whitney U-test, P=0.0005). In addition to the mature fibulin-1 polypeptide, several smaller sized polypeptides of 55, 50 and 25 kDa were detected using monoclonal antibodies reactive towards an epitope located at the N-terminus of fibulin-1. The immunoreactive 50 kDa polypeptide was detected more frequently in breast carcinoma specimens than in normal breast tissue (chi(2)=17.22, P<0.0001). Furthermore, the ratio of the 50 kDa fragment to the mature fibulin-1 polypeptide correlated with the level of oestrogen receptor alpha (Spearman correlation coefficient, rs=0.49, P<0.003, n=36) and progesterone receptor (rs=0.43, P=0.008, n=36) expression in the tumour specimens. Taken together, these findings indicate that elevated expression and altered processing of fibulin-1 is associated with human breast cancer.

Antibodies, Monoclonal↗

PubMed2002

The fibulin-1 gene (FBLN1) is disrupted in a t(12;22) associated with a complex type of synpolydactyly.

Molecular analysis of the reciprocal chromosomal translocation t(12;22)(p11.2;q13.3) cosegregating with a complex type of synpolydactyly showed involvement of an alternatively spliced exon of the fibulin-1 gene (FBLN1 located in 22q13.3) and the C12orf2 (HoJ-1) gene on the short arm of chromosome 12. Investigation of the possible functional involvement of the fibulin-1 protein (FBLN1) in the observed phenotype showed that FBLN1 is expressed in the extracellular matrix (ECM) in association with the digits in the developing limb. Furthermore, fibroblasts derived from patients with the complex type of synpolydactyly displayed alterations in the level of FBLN1-D splice variant incorporated into the ECM and secreted into the conditioned culture medium. By contrast, the expression of the FBLN1-C splice variant was not perturbed in the patient fibroblasts. Based on these findings, we propose that the t(12;22) results in haploinsufficiency of the FBLN1-D variant, which could lead to the observed limb malformations.

Animals↗

PubMed2001

Members of the low density lipoprotein receptor family control diverse physiological processes.

The low density lipoprotein receptor (LDLR) family is a group of receptors that mediate endocytosis leading to lysosomal degradation of an enormous repertoire of ligands. To date, over 50 distinct macromolecules have been shown to bind members of the family. This wide spectrum of ligand recognition is the basis for an immense diversity in physiological processes in which these receptors participate. In this article, the physiological roles of the LDLR family members are briefly reviewed and a comprehensive list of the ligands with which the receptors interact is presented.

Absorption↗

PubMed2001

Cubilin and megalin: partners in lipoprotein and vitamin metabolism.

Members of the low-density lipoprotein receptor (LDLR) family are unrivalled for their ability to endocytose and target ligands to lysosomes for degradation. Their endocytic and catabolic functions make them essential to homeostatic regulation of the level and activity of their ligands in biological fluids and interstitial spaces. Over the last few years it has become evident that the endocytic function of members of the LDLR family is employed by other kinds of cell surface receptors. Recently, the low-density lipoprotein receptor related protein-2 (megalin) was shown to act in concert with cubilin, a receptor for high-density lipoproteins (HDL)/apolipoprotein A-I (apoA-I), intrinsic factor-vitamin B12 and albumin to mediate ligand endocytosis. In this article, we review the state of knowledge pertaining to cubilin and megalin, emphasizing their joint roles in both lipoprotein and vitamin metabolism.

Animals↗

PubMed2001

Fibulin-1 suppression of fibronectin-regulated cell adhesion and motility.

Fibulin-1 is an extracellular matrix protein often associated with fibronectin (FN) in vivo. In this study, the ability of fibulin-1 to modulate adhesion, spreading and motility-promoting activities of FN was investigated. Fibulin-1 was found to have pronounced inhibitory effects on the cell attachment and spreading promoted by FN. Fibulin-1 was also found to inhibit the motility of a variety of cell types on FN substrata. For example, the FN-dependent haptotactic motility of breast carcinoma (MDA MB231) cells, epidermal carcinoma (A431), melanoma (A375 SM), rat pulmonary aortic smooth muscle cells (PAC1) and Chinese hamster ovary (CHO) cells was inhibited by the presence of fibulin-1 bound to FN-coated Boyden chamber membranes. Cells transfected to overproduce fibulin-1 displayed reduced velocity, distance of movement and persistence time on FN substrata. Similarly, the incorporation of fibulin-1 into FN-containing type I collagen gels inhibited the invasion of endocardial cushion mesenchymal cells migrating from cultured embryonic heart explants. By contrast, incorporation of fibulin-1 into collagen gels lacking FN had no effect on the migration of endocardial cushion cells. These results suggest that the motility-suppressive effects of fibulin-1 might be FN specific. Furthermore, such effects are cell-type specific, in that the migration of gingival fibroblasts and endothelial cells on FN substrata is not responsive to fibulin-1. Additional studies found that the mechanism for the motility-suppressive effects of fibulin-1 does not involve perturbations of interactions between alpha5beta1 or alpha4 integrins, or heparan sulfate proteoglycans with FN. However, fibulin-1 was found to inhibit extracellular signal regulated kinase (ERK) activation and to suppress phosphorylation of myosin heavy chain. This ability to influence signal transduction cascades that modulate the actin-myosin motor complex might be the basis for the effects of fibulin-1 on adhesion and motility.

Actomyosin↗

PubMed2000

Megalin acts in concert with cubilin to mediate endocytosis of high density lipoproteins.

Cubilin has recently been shown to function as an endocytic receptor for high density lipoproteins (HDL). The lack of apparent transmembrane and cytoplasmic domains in cubilin raises questions as to the means by which it can mediate endocytosis. Since cubilin has been reported to bind the endocytic receptor megalin, we explored the possibility that megalin acts in conjunction with cubilin to mediate HDL endocytosis. While megalin did not bind to HDL, delipidated HDL, or apoA-I, it was found to copurify with cubilin isolated by HDL-Sepharose affinity chromatography. Cubilin and megalin exhibited coincident patterns of mRNA expression in mouse tissues including the kidney, ileum, thymus, placenta, and yolk sac endoderm. The expression of both receptors in yolk sac endoderm-like cells was inducible by retinoic acid treatment but not by conditions of sterol depletion. Suppression of megalin activity or expression by treatment with either megalin antibodies or megalin antisense oligodeoxynucleotides resulted in inhibition of cubilin-mediated endocytosis of HDL. Furthermore, megalin antisense oligodeoxynucleotide treatment resulted in reduced cell surface expression of cubilin. These data demonstrate that megalin acts together with cubilin to mediate HDL endocytosis and further suggest that megalin may play a role in the intracellular trafficking of cubilin.

Animals↗

PubMed1999

Cubilin, the endocytic receptor for intrinsic factor-vitamin B(12) complex, mediates high-density lipoprotein holoparticle endocytosis.

Receptors that endocytose high-density lipoproteins (HDL) have been elusive. Here yolk-sac endoderm-like cells were used to identify an endocytic receptor for HDL. The receptor was isolated by HDL affinity chromatography and identified as cubilin, the recently described endocytic receptor for intrinsic factor-vitamin B(12). Cubilin antibodies inhibit HDL endocytosis by the endoderm-like cells and in mouse embryo yolk-sac endoderm, a prominent site of cubilin expression. Cubilin-mediated HDL endocytosis is inhibitable by HDL(2), HDL(3), apolipoprotein (apo)A-I, apoA-II, apoE, and RAP, but not by low-density lipoprotein (LDL), oxidized LDL, VLDL, apoC-I, apoC-III, or heparin. These findings, coupled with the fact that cubilin is expressed in kidney proximal tubules, suggest a role for this receptor in embryonic acquisition of maternal HDL and renal catabolism of filterable forms of HDL.

Animals↗

PubMed1999

Identification of low density lipoprotein receptor-related protein-2/megalin as an endocytic receptor for seminal vesicle secretory protein II.

The low density lipoprotein receptor-related protein-2/megalin (LRP-2) is an endocytic receptor that is expressed on the apical surfaces of epithelial cells lining specific regions of the male and female reproductive tracts. In the present study, immunohistochemical staining revealed that LRP-2 is also expressed by epithelial cells lining the ductal region and the ampulla of the rat seminal vesicle. To identify LRP-2 ligands in the seminal vesicle, we probed seminal vesicle fluid with 125I-labeled LRP-2 in a gel-blot overlay assay. A 100-kDa protein (under non-reducing conditions) was found to bind the radiolabeled receptor. The protein was isolated and subjected to protease digestion, and the proteolytic fragments were subjected to mass spectroscopic sequence analysis. As a result, the 100-kDa protein was identified as the seminal vesicle secretory protein II (SVS-II), a major constituent of the seminal coagulum. Using purified preparations of SVS-II and LRP-2, solid-phase binding assays were used to show that the SVS-II bound to the receptor with high affinity (Kd = 5.6 nM). The binding of SVS-II to LRP-2 was inhibited using a known antagonist of LRP-2 function, the 39-kDa receptor-associated protein RAP. Using a series of recombinant subfragments of SVS-II, the LRP-2 binding site was mapped to a stretch of repeated 13-residue modules located in the central portion of the SVS-II polypeptide. To evaluate the ability of LRP-2 to mediate 125I-SVS-II endocytosis and lysosomal degradation, ligand clearance assays were performed using differentiated mouse F9 cells, which express high levels of LRP-2. Radiolabeled SVS-II was internalized and degraded by the cells, and both processes were inhibited by antibodies to LRP-2 or by RAP. The results indicate that LRP-2 binds SVS-II and can mediate its endocytosis leading to lysosomal degradation.

Amino Acid Sequence↗

PubMed1999

Expression and regulation of LRP-2/megalin in epithelial cells lining the efferent ducts and epididymis during postnatal development.

Low density lipoprotein receptor-related protein-2/megalin (LRP-2) is a receptor belonging to the low density lipoprotein receptor family that mediates endocytosis and lysosomal degradation of a variety of ligands including apolipoprotein J (Apo J)/clusterin/SGP-2. LRP-2 has been shown to be expressed regionally in the adult rat epididymis. In this study, we describe the pattern of expression of LRP-2 in the efferent ducts and epididymis during postnatal development of the rat and examine the role of testicular luminally derived substances on its expression. The expression of LRP-2 was analyzed immunocytochemically in tissues of normal animals ranging in age from postnatal day 7-90 and in 15-day-old efferent-duct-ligated animals sacrificed at later ages. In the efferent ducts, LRP-2 expression, appearing as a dense band on the apical surface of the nonciliated epithelial cells, was noted as early as day 7, well before the entry of sperm, Sertoli-cell-derived secretory products, and high levels of androgens. Efferent duct ligation studies further revealed that expression under this condition was comparable to controls at all later ages examined, suggesting that the factor regulating its expression was not a luminally derived testicular substance. In normal untreated animals, LRP-2 expression was not apparent at any of the ages examined in the proximal initial segment of the epididymis. By comparison, the distal initial segment, although having no LRP-2 expression from 7-15 days, showed expression in principal cells by day 21 which intensified at days 29 and 39. However, by day 49 and at later ages (56 and 90), LRP-2 immunoreactivity over principal cells became spotty or with weak or moderate reactivity in some cells and none in others. LRP-2 expression in the intermediate zone, proximal caput, corpus, and cauda regions also appeared in principal cells by day 21, intensified at days 29 and 39 and persisted as such at all later ages examined, correlating with high levels of androgens shown to occur by day 39. Although LRP-2 expression in the distal caput region was evident in principal cells at days 21 and 29, it became spotty with weak, moderate, or absent reactivity over principal cells at all later ages. These data suggest that LRP-2 expression is under the influence of both stimulatory and region-specific inhibitory factors. Analysis of 15-day-old efferent-duct-ligated animals at all later ages examined revealed that there was no change in LRP-2 expression along the entire epididymis, suggesting that both the stimulatory and inhibitory factors are not luminally derived testicular substances. The observed pattern of LRP-2 expression in all regions of the epididymis, except the distal caput region, was similar to that described for Apo J internalization by principal cells during postnatal development, showing a correlation between LRP-2 expression and its ligand, Apo J. In summary, LRP-2 expression in the epididymis undergoes region-specific changes during postnatal development and appears to be influenced by both stimulatory and inhibitory factors.

Animals↗

PubMed1998

Estradiol and fibulin-1 inhibit motility of human ovarian- and breast-cancer cells induced by fibronectin.

Ovarian-cancer cells are characterized by their ability to invade freely the peritoneal cavity. Estradiol stimulates the proliferation of estrogen-receptor(ER)-positive ovarian-cancer cells, as well as expression of fibulin-1, a fibronectin-binding extracellular matrix protein. Using a modified Boyden-chamber assay, we have evaluated the respective roles of estradiol and fibulin-1 on cell motility, one of the earlier steps of tumor invasion. The effect of estradiol was examined on the random and directional migration of different ER-positive ovarian-cancer cell lines. The effect of fibulin-1 was studied on the motility of the MDA-MB231 breast-cancer cell line, which does not express fibulin-1. We found that when fibronectin (FN) was used as an attractant, estradiol decreased the cell motility of 2 ER-positive ovarian-cancer cell lines, BG-1 and SKOV3, but had no effect on 2 ER-negative cell lines, PEO14 and MDA-MB231. The inhibitory effect of estradiol was not observed when collagen (type 1 or 4) or laminin were used as attractants. Fibulin-1 was found to inhibit haptotactic migration of MDA-MB231 cells to FN in a dose-dependent manner. We conclude that both estradiol and fibulin-1 inhibit cancer cell motility in vitro and therefore have the potential to inhibit tumor invasion.

Breast Neoplasms↗

PubMed1998

Identification of the developmental marker, JB3-antigen, as fibrillin-2 and its de novo organization into embryonic microfibrous arrays.

The monoclonal antibody JB3 was previously shown to react with a protein antigen present in the bilateral primitive heart-forming regions and septation-stage embryonic hearts; in addition, primary axial structures at primitive streak stages are JB3-immunopositive (Wunsch et al. [1994] Dev. Biol. 165:585-601). The JB3 antigen has an overlapping distribution pattern with fibrillin-1, and a similar molecular mass (Gallagher et al. [1993] Dev. Dyn. 196:70-78; Wunsch et al. [1994] Dev. Biol. 165:585-601). Here we present immunoblot and immunoprecipitation data showing that the JB3 antigen is secreted into tissue culture medium by day 10 chicken embryonic fibroblasts, from which it can be harvested using JB3-immunoaffinity chromatography. A single polypeptide (Mr = 350,000), which was not immunoreactive with an antibody to fibrillin-1, eluted from the affinity column. Mass spectroscopy peptide microsequencing determined the identity of the JB3 antigen to be an avian homologue of fibrillin-2. Live, whole-mounted, quail embryos were immunolabeled using a novel microinjection approach, and subsequently fixed. Laser scanning confocal microscopy indicated an elaborate scaffold of fibrillin-2 filaments encasing formed somites. At more caudal axial positions, discrete, punctate foci of immunofluorescent fibrillin-2 were observed; this pattern corresponded to the position of segmental plate mesoderm. Between segmental plate mesoderm and fully-formed somites, progressively longer filamentous assemblies of fibrillin-2 were observed, suggesting a developmental progression of fibrillin-2 fibril assembly across the somite-forming region of avian embryos. Extensive filaments of fibrillin-2 connect somites to the notochord. Similarly, fibrillin-2 connects the mesoderm associated with the anterior intestinal portal to the midline. Thus, fibrillin-2 fibrils are organized by a diverse group of cells of mesodermal or mesodermally derived mesenchymal origin. Fibrillin-2 microfilaments are assembled in a temporal and spatial pattern that is coincident with cranial-to-caudal segmentation, and regression of the anterior intestinal portal. Fibrillin-2 may function to impart physical stability to embryonic tissues during morphogenesis of the basic vertebrate body plan.

Actin Cytoskeleton↗

PubMed1998

Identification of chicken and C. elegans fibulin-1 homologs and characterization of the C. elegans fibulin-1 gene.

Fibulin-1, a member of the emerging family of fibulin proteins, is a component of elastic extracellular matrix fibers, basement membranes and blood. Homologs of fibulin-1 have been described in man, mouse and zebrafish. In this study, we describe the isolation and sequencing of chicken fibulin-1C and D cDNA variants. We also describe identification of a C. elegans cDNA encoding fibulin-1D and cosmids containing the C. elegans fibulin-1 gene. Using the cDNA, RT-PCR and computer-based analysis of genomic sequences, the exon/intron organization of the C. elegans fibulin-1 gene was determined. The C. elegans fibulin-1 gene is located on chromosome IV, is approximately 6 kb in length, contains 16 exons and encodes fibulin-1C and D variants. Comparative analysis of the deduced amino acid sequences of nematode and chicken fibulin-1 variants with other known vertebrate fibulin-1 polypeptides showed that the number and organization of structural modules are identical. The results of this study indicate that the structure of the fibulin-1 protein has remained highly conserved over a large period of evolution, suggestive of functional conservation.

Amino Acid Sequence↗

PubMed1997

The self-association and fibronectin-binding sites of fibulin-1 map to calcium-binding epidermal growth factor-like domains.

Fibulin-1 is a modular glycoprotein with amino-terminal anaphylatoxin-like modules followed by nine epidermal growth factor (EGF)-like modules and, depending on alternative splicing, four possible carboxyl termini. Fibulin-1 has been shown to self-associate as well as to bind calcium, fibronectin (FN), laminin, nidogen, and fibrinogen. To map ligand-binding sites within fibulin-1, polypeptides corresponding to various regions of fibulin-1 were expressed recombinantly and evaluated for their capacity to bind calcium, FN, or fibulin-1. A calcium-binding site(s) was mapped to EGF-like modules 5-9. A fibulin-1 self-association site was localized to EGF-like modules 5 and 6 (amino acid residues 356-440), as was a binding site for FN. The self-association interaction mediated by this pair of modules involved calcium since divalent cation chelators reduced the binding affinity of the interaction. By contrast, FN binding to EGF-like modules 5 and 6 was unaffected by the presence of divalent cation chelators. It can be concluded that EGF-like modules 5 and 6 bind calcium and mediate homotypic interaction between EGF-like modules 5 and 6 present in different fibulin-1 molecules and heterotypic interaction between EGF-like modules 5 and 6 and type III repeats 13 and 14 in FN. While additional binding sites for calcium or FN were not detected, another fibulin-1 self-association site was found within amino acid residues 30-173. However, unlike the self-association site in EGF-like modules 5 and 6, which was functional in the native protein, the amino-terminal site was cryptic and revealed only after the protein was denatured.

Binding Sites↗

PubMed1997

Interaction of apolipoprotein J-amyloid beta-peptide complex with low density lipoprotein receptor-related protein-2/megalin. A mechanism to prevent pathological accumulation of amyloid beta-peptide.

Apolipoprotein J (apoJ) has been shown to be the predominant amyloid beta-peptide (Abeta)-binding protein in cerebrospinal fluid. We have previously demonstrated that the endocytic receptor low density lipoprotein receptor-related protein-2/megalin (LRP-2), which is expressed by choroid plexus epithelium and ependymal cells lining the brain ventricles and neural tube, binds and mediates cellular uptake of apoJ (Kounnas, M. Z., Loukinova, E. B., Stefansson, S., Harmony, J. A., Brewer, B., Strickland, D. K., and Argraves, W. S. (1995) J. Biol. Chem. 270, 13070-13075). In the present study, we evaluated the ability of apoJ to mediate binding of Abeta1-40-apoJ complex to LRP-2 in vitro. Immunoblot analysis showed that incubation of apoJ with Abeta1-40 resulted in the formation of Abeta1-40-apoJ complex and the inhibition of the formation of Abeta1-40 aggregates. Using an enzyme-linked immunosorbent assay, an estimated dissociation constant (Kd) of 4.8 nM was derived for the interaction between Abeta1-40 and apoJ. Enzyme-linked immunosorbent assay was also used to study the interaction of the Abeta1-40-apoJ complex with LRP-2. The results showed that Abeta alone did not bind directly to LRP-2; however, when Abeta1-40 was combined with apoJ to form a complex, binding to LRP-2 took place. The binding interaction could be blocked by inclusion of the receptor-associated protein, an antagonist of apoJ binding to LRP-2. When LRP-2-expressing cells were given 125I-Abeta1-40, cellular uptake of the radiolabeled peptide was promoted by co-incubation with apoJ. When the cells were provided purified 125I-Abeta1-40-apoJ complex, the complex was internalized and degraded, and both processes were inhibited with polyclonal LRP-2 antibodies. Furthermore, chloroquine treatment inhibited the cellular degradation of the complex. The data indicate that apoJ facilitates Abeta1-40 binding to LRP-2 and that the receptor mediates cellular clearance of Abeta1-40-apoJ complex leading to lysosomal degradation of Abeta1-40. The findings support the possibility that LRP-2 can act in vivo to mediate clearance of the complex from biological fluids such as cerebrospinal fluid and thereby play a role in the regulation of Abeta accumulation.

Amyloid beta-Peptides↗

PubMed1997

Identification of the 170-kDa melanoma membrane-bound gelatinase (seprase) as a serine integral membrane protease.

The 170-kDa membrane-bound gelatinase, seprase, is a cell surface protease, the expression of which correlates with the invasive phenotype of human melanoma and carcinoma cells. We have isolated seprase from cell membranes and shed vesicles of LOX human melanoma cells. The active enzyme is a dimer of N-glycosylated 97-kDa subunits. Sequence analysis of three internal proteolytic fragments of the 97-kDa polypeptide revealed up to 87.5% identity to the 95-kDa fibroblast activation protein alpha (FAPalpha), the function of which is unknown. Thus, we used reverse transcription-polymerase chain reaction to generate a 2.4-kilobase cDNA from LOX mRNA with FAPalpha primers. COS-7 cells transfected with this cDNA expressed a 170-kDa gelatinase that is recognized by monoclonal antibodies directed against seprase. Sequence analysis also showed similarities to the 110-kDa subunit of dipeptidyl peptidase IV (DPPIV). Like DPPIV, the gelatinase activity of seprase was completely blocked by serine-protease inhibitors, including diisopropyl fluorophosphate. Seprase could be affinity-labeled by [3H]diisopropyl fluorophosphate, but the proteolytically inactive 97-kDa subunit could not, confirming the existence of a serine protease active site on the dimeric form. Proteolytic activity is lost upon dissociation into its 97-kDa subunit following treatment with acid, heat, or cysteine and histidine-modifying agents. We conclude that seprase, FAPalpha, and DPPIV are related serine integral membrane proteases and that seprase is similar to DPPIV, the proteolytic activities of which are dependent upon subunit association.

Amino Acid Sequence↗

PubMed1997

Human fibulin-1D: molecular cloning, expression and similarity with S1-5 protein, a new member of the fibulin gene family.

Fibulin-1 is an extracellular matrix (ECM) component of basement membranes and connective tissue elastic fibers, and a blood protein. Multiple forms of fibulin-1 that differ in their C-terminal regions are produced through the process of alternative splicing of their precursor RNA. Two transcripts of 2.4 and 2.7 kb are the predominant fibulin-1 mRNAs expressed in human tissues and cultured cells. While the 2.4 kb transcript had been shown to encode fibulin-1C, the 2.7 kb transcript did not correspond to any of the previously identified human fibulin-1 variants. Herein, we report on the isolation and sequencing of cDNA corresponding to the 2.7 kb fibulin-1 transcript which encodes a novel, alternatively spliced form of human fibulin-1 that we term the D form. The deduced amino acid sequence of the D form is identical in its first 566 residues to the three known fibulin-1 variants (fibulin-1A-C); however, it has a unique 137 amino acid-C-terminal segment encoded by the alternatively spliced portion of its transcript. RNA hybridization analysis showed that the fibulin-1D transcript is coordinately expressed with that of fibulin-1C both in tissues and in cultured cells. Using antibodies specific to the unique C-terminal segment of fibulin-1D and -1C, both proteins were found to be expressed in human placenta. Recombinant fibulin-1D generated in transfected mammalian cells displayed similar ligand-binding properties as placenta-derived fibulin-1 and recombinant fibulin-1C, and it was capable of incorporating into cultured cell ECM in the absence of other fibulin-1 forms. A comparative sequence analysis revealed that the unique C-terminal region of fibulin-1D is similar to the C-terminal regions of fibulin-1C and fibulin-2. Furthermore, the C-terminal regions of fibulin-1C, -1D and -2 are similar to the C-terminal region of a recently described protein termed S1-5. In addition to this C-terminal similarity, S1-5 also contains repeated EGF-like modules and a conserved N-terminal element, thereby leading to the conclusion that S1-5 is a third member of the fibulin gene family.

Amino Acid Sequence↗

PubMed1997

Suppression of anchorage-independent growth and matrigel invasion and delayed tumor formation by elevated expression of fibulin-1D in human fibrosarcoma-derived cell lines.

Using differential display, we identified an mRNA that is markedly down-regulated in cell line 6A/SB1, derived from a fibrosarcoma formed in an athymic mouse following injection of carcinogen-transformed MSU-1.1 cells. The nontumorigenic parental cell strain, MSU-1.1, expresses high levels of this mRNA. Sequencing of the corresponding cDNA fragment revealed that it corresponded to an expressed sequence tag, which ultimately led to its identification as the fibulin-1D gene. Fibulin-1 is a cysteine-rich, calcium-binding extracellular matrix and plasma protein, which has four isoforms, A-D, derived from alternative splicing. Northern and Western blotting analysis of 16 cell lines established from tumors formed in athymic mice by MSU-1.1-derived cell strains independently transformed in culture showed that 44% exhibited low level or lack of expression of fibulin-1D mRNA and protein. In a similar analysis of 15 malignant cell lines derived from patients, 80% showed low level or no expression. To study the role of fibulin-1D in transformation, we transfected 6A/SB1 cells and a human fibrosarcoma-derived cell line (SHAC) with a fibulin-1D cDNA expression construct. Transfectants displaying high levels of fibulin-1D were isolated and characterized. Elevated expression of fibulin-1D led to reduced ability to form colonies in soft agar and reduced invasive potential as tested in a matrigel in vitro invasion assay. Furthermore, expression of fibulin-1D resulted in a markedly extended latency in tumor formation in athymic mice. These results indicate that low expression of fibulin-1D plays a role in tumor formation and invasion.

Animals↗