Basement Membrane Protein Nidogen Research Articles

Human Cytomegalovirus Utilizes Multiple Viral Proteins to Regulate the Basement Membrane Protein Nidogen 1.

Nidogen 1 (NID1) is an important basement membrane protein secreted by many cell types. We previously found that human cytomegalovirus (HCMV) infection rapidly induced chromosome 1 breaks and that the basement membrane protein NID1, encoded near the 1q42 break site, was downregulated. We have now determined that the specific breaks in and of themselves did not regulate NID1, rather interactions between several viral proteins and the cellular machinery and DNA regulated NID1. We screened a battery of viral proteins present by 24 hours postinfection (hpi) when regulation was induced, including components of the incoming virion and immediate early (IE) proteins. Adenovirus (Ad) delivery of the tegument proteins pp71 and UL35 and the IE protein IE1 influenced steady-state (ss) NID1 levels. IE1's mechanism of regulation was unclear, while UL35 influenced proteasomal regulation of ss NID1. Real-time quantitative PCR (RT-qPCR) experiments determined that pp71 downregulated NID1 transcription. Surprisingly, WF28-71, a fibroblast clone that expresses minute quantities of pp71, suppressed NID1 transcription as efficiently as HCMV infection, resulting in the near absence of ss NID1. Sequence analysis of the region surrounding the 1q42 break sites and NID1 promoter revealed CCCTC-binding factor (CTCF) binding sites. Chromatin immunoprecipitation experiments determined that pp71 and CTCF were both bound at these two sites during HCMV infection. Expression of pp71 alone replicated this binding. Binding was observed as early as 1 hpi, and colocalization of pp71 and CTCF occurred as quickly as 15 min postinfection (pi) in infected cell nuclei. In fibroblasts where CTCF was knocked down, Adpp71 infection did not decrease NID1 transcription nor ss NID1 protein levels. Our results emphasize another aspect of pp71 activity during infection and identify this viral protein as a key contributor to HCMV's efforts to eliminate NID1. Further, we show, for the first time, direct interaction between pp71 and the cellular genome. IMPORTANCE We have found that human cytomegalovirus (HCMV) utilizes multiple viral proteins in multiple pathways to regulate a ubiquitous cellular basement membrane protein, nidogen-1 (NID1). The extent of the resources and the redundant methods that the virus has evolved to affect this control strongly suggest that its removal provides a life cycle advantage to HCMV. Our discoveries that one of the proteins that HCMV uses to control NID1, pp71, binds directly to the cellular DNA and can exert control when present in vanishingly small quantities may have broad implications in a wide range of infection scenarios. Dysregulation of NID1 in an immunocompetent host is not known to manifest complications during infection; however, in the naive immune system of a developing fetus, disruption of this developmentally critical protein could initiate catastrophic HCMV-induced birth defects.

Human Cytomegalovirus Interactions with the Basement Membrane Protein Nidogen 1.

In 2000, we reported that human cytomegalovirus (HCMV) induced specific damage on chromosome 1. The capacity of the virus to induce DNA breaks indicated potent interaction between viral proteins and these loci. We have fine mapped the 1q42 breaksite. Transcriptional analysis of genes encoded in close proximity revealed virus-induced downregulation of a single gene, nidogen 1 (NID1). Beginning between 12 and 24 hours postinfection (hpi) and continuing throughout infection, steady-state (ss) NID1 protein levels were decreased in whole-cell lysates and secreted supernatants of human foreskin fibroblasts. Addition of the proteasomal inhibitor MG132 to culture medium stabilized NID1 in virus-infected cells, implicating infection-activated proteasomal degradation of NID1. Targeting of NID1 via two separate pathways highlighted the virus' emphasis on NID1 elimination. NID1 is an important basement membrane protein secreted by many cell types, including the endothelial cells (ECs) lining the vasculature. We found that ss NID1 was also reduced in infected ECs and hypothesized that virus-induced removal of NID1 might offer HCMV a means of increased distribution throughout the host. Supporting this idea, transmigration assays of THP-1 cells seeded onto NID1-knockout (KO) EC monolayers demonstrated increased transmigration. NID1 is expressed widely in the developing fetal central and peripheral nervous systems (CNS and PNS) and is important for neuronal migration and neural network excitability and plasticity and regulates Schwann cell proliferation, migration, and myelin production. We found that NID1 expression was dramatically decreased in clinical samples of infected temporal bones. While potentially beneficial for virus dissemination, HCMV-induced elimination of NID1 may underlie negative ramifications to the infected fetus.IMPORTANCE We have found that HCMV infection promotes the elimination of the developmentally important basement membrane protein nidogen 1 (NID1) from its host. The virus both decreased transcription and induced degradation of expressed protein. Endothelial cell (EC) secretion of basement membrane proteins is critical for vascular wall integrity, and infection equivalently affected NID1 protein levels in these cells. We found that the absence of NID1 in an EC monolayer allowed increased transmigration of monocytes equivalent to that observed after infection of ECs. The importance of NID1 in development has been well documented. We found that NID1 protein was dramatically reduced in infected inner ear clinical samples. We believe that HCMV's attack on host NID1 favors viral dissemination at the cost of negative developmental ramifications in the infected fetus.

BMI-1 Promotes Ewing Sarcoma Tumorigenicity Independent ofCDKN2ARepression

Deregulation of the polycomb group gene BMI-1 is implicated in the pathogenesis of many human cancers. In this study, we have investigated if the Ewing sarcoma family of tumors (ESFT) expresses BMI-1 and whether it functions as an oncogene in this highly aggressive group of bone and soft tissue tumors. Our data show that BMI-1 is highly expressed by ESFT cells and that, although it does not significantly affect proliferation or survival, BMI-1 actively promotes anchorage-independent growth in vitro and tumorigenicity in vivo. Moreover, we find that BMI-1 promotes the tumorigenicity of both p16 wild-type and p16-null cell lines, demonstrating that the mechanism of BMI-1 oncogenic function in ESFT is, at least in part, independent of CDKN2A repression. Expression profiling studies of ESFT cells following BMI-1 knockdown reveal that BMI-1 regulates the expression of hundreds of downstream target genes including, in particular, genes involved in both differentiation and development as well as cell-cell and cell-matrix adhesion. Gain and loss of function assays confirm that BMI-1 represses the expression of the adhesion-associated basement membrane protein nidogen 1. In addition, although BMI-1 promotes ESFT adhesion, nidogen 1 inhibits cellular adhesion in vitro. Together, these data support a pivotal role for BMI-1 ESFT pathogenesis and suggest that its oncogenic function in these tumors is in part mediated through modulation of adhesion pathways.

Identification of the basement membrane protein nidogen as a candidate ligand for tumor endothelial marker 7 in vitro and in vivo

Tumor endothelial marker 7 (TEM7) is a novel transmembrane protein that is highly expressed in the tumor endothelium. However, the ligands and functions of TEM7 are unknown at present. Using a recombinant ectodomain of TEM7 as a probe, we could identify the saturable interaction between nidogen and TEM7 in vitro. A cell overlay binding assay showed that recombinant nidogen interact with full length TEM7 on cell surface. Finally, nidogen/TEM7 interaction enhanced cell spreading in TEM7 transfected 293T cells. This novel nidogen/TEM7 interaction may provide an important molecular tool with potential therapeutic applications in the anti-angiogenic therapies.

Complete exon-intron organization of the mouse fibulin-1 gene and its comparison with the human fibulin-1 gene

Fibulin-1 is a 90 kDa calcium-binding protein present in the extracellular matrix and in the blood. Two major variants, C and D, differ in their C-termini as well as the ability to bind the basement membrane protein nidogen. Here we characterized genomic clones encoding the mouse fibulin-1 gene, which contains 18 exons spanning at least 75 kb of DNA. The two variants are generated by alternative splicing of exons in the 3′ end. By searching the database we identified most of the exons encoding the human fibulin-1 gene and showed that its exon-intron organization is similar to that of the mouse gene.

Structural Characterization of Two Variants of Fibulin-1 that Differ in Nidogen Affinity

Two C-terminal variants C and D of mouse fibulin-1 were purified from the culture medium of stably transfected human kidney cell clones. They showed, after rotary shadowing, a dumbbell-like structure of about 33 nm in length. Pepsin digestion demonstrated stability of the disulfide-bonded domains I (anaphylatoxin-like) and II (multiple EGF-like motifs) but not for domain III which is different in the variants. A close similarity of the variants was observed in immunochemical assays indicating that domain III epitopes are not very antigenic. Binding analysis in solid phase assays demonstrated for variant C a 100-fold stronger binding to the basement membrane protein nidogen than for variant D. Both interactions were sensitive to EDTA. Surface plasmon resonance assays confirmed this difference and showed K D=60 nM for variant C and K D→1 μM for variant D. Lower binding activities and smaller differences between both variants were observed for the calcium-dependent binding to fibronectin, laminin-1 and collagen IV. Self aggregation into nest-like oligomers was observed at high concentrations of fibulin-1 which was not sensitive to EDTA.

Structure and Localization of O- and N-Linked Oligosaccharide Chains on Basement Membrane Protein Nidogen

The carbohydrate content of mouse nidogen predicts the occupation of two N- and about seven O-linked acceptor sites. The corresponding oligosaccharides were examined by sequential exoglycosidase digestions. The data indicate N-linked substitutions by several bi-, tri- and tetraantennary complex types of oligosaccharides which are further modified by additional lactosamines and terminal α-galactose and/or sialic acid. Mannose-rich oligosaccharides were of low abundance. O-linked structures included a di- and tetrasaccharide core structure that were in addition sialylated and may be similar to structures found in fetuin. Evidence is provided that the two sequence-predicted asparagine acceptors are almost fully substituted. Sequence analysis of tryptic peptides identified Thr-271, Ser-303, Thr-309, Thr-317, Thr-320, Thr-892 and Thr-905 as the most likely sites for galactosamine substitutions. These residues are located in the flexible link connecting the N-terminal globular domains G1 and G2 of nidogen and at the border between the rod and the C-terminal globe G3. Four of them showed Pro in the −1 or + 3 position. All these Ser, Thr and Pro residues but not the N-linked attachment sites are identical in human nidogen.

Antibodies to basement membrane protein nidogen in Chagas' disease and American cutaneous leishmaniasis

About 50 to 70% of sera from patients with American cutaneous leishmaniasis and chronic Chagas' disease possessed antibodies which reacted in enzyme and radioimmunoassays with nidogen obtained from a tumor basement membrane. The antibodies were of the immunoglobulin M and G classes in acute American cutaneous leishmaniasis but mainly of the immunoglobulin G class in chronic Chagas' disease. Similar antibodies could not be detected in patients suffering from a variety of other infectious or inflammatory diseases when compared with healthy control groups. Inhibition and immunoadsorption studies indicated a close relationship of epitopes recognized by patients' antibodies on nidogen and on another basement membrane protein, laminin. Since rabbit antisera to both proteins do not cross-react, a special nature of the epitopes involved in the reaction with patient sera is suggested. Similar epitopes may exist on various forms of Leishmania or Trypanosoma protozoa.

Identification and interaction repertoire of large forms of the basement membrane protein nidogen.

Nidogen was purified in its genuine form with a mol. wt. of 150 000 (Nd-150) and as fragments with mol. wts. of 100 000 (Nd-100) and 80 000 (Nd-80) from a mouse tumor basement membrane by preventing activity of endogenous proteases with 6 M guanidine and protease inhibitors. The larger forms of nidogen were also identified in stable complexes with laminin in neutral salt extracts of the tumor and in cell culture medium. Purified Nd-150 and Nd-100, but not Nd-80, were shown to interact with laminin in various binding assays, albeit with lower potential than estimated for the genuine complexes formed in situ. Binding of Nd-150 and Nd-100 to fibronectin and to the globular domain of collagen IV was also observed, but not to heparan sulfate proteoglycan.