Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for approximately 25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.     

Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex

We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.     

A widely distributed nuclear protein immunologically related to the microtubule-associated protein MAP1 is associated with the mitotic spindle.

A 280-kDa protein (p280) confined to the nucleus of interphase cells becomes associated with the mitotic spindle during cell division. p280 is immunologically related to the microtubule-associated protein MAP1, as shown by cross-reactivity with monoclonal (8D12) and polyclonal antibodies raised against MAP1. However, p280 is distinct from MAP1 as judged by its lower molecular size, proteolytic degradation products, presence in preparations of purified nuclei from which MAP1 is absent, and absence from the cytosol fraction that contains MAP1. Immunofluorescence microscopy of cells in interphase using 8D12 reveals punctate staining of the nucleus, cytoplasmic microtubules, and the microtubule organizing center. Dividing cells display strong staining of the spindle, centrioles, and mid-body. The only exception to this staining pattern is marsupial Pt k2 cells that contain p280 in the nucleus and lack MAP1. These cells exhibit fluorescent staining of the nucleus and the microtubule organizing center when in interphase, of spindle and centrioles in mitosis, and show no staining of cytoplasmic and mid-body microtubules.     

Glucose transport protein is structurally and immunologically related to band 3 and senescent cell antigen.

Senescent cell antigen, a polypeptide that appears on the surface of senescent and damaged cells, has been shown to be derived from band 3. In the present study, the relationship between the as yet unidentified glucose transporter and senescent cell antigen is examined. Since cytochalasin B is a specific and potent competitive inhibitor of glucose transport in human erythrocytes, the glucose transport carrier was isolated by affinity chromatography on cytochalasin B-Sepharose 4B columns and eluted with D-glucose. This purification procedure is both a method of isolation and a functional assay for the glucose transporter. Purified glucose transporter gave a sharp, single band at Mr approximately equal to 60,000 when analyzed by NaDod-SO4/PAGE. Glucose transporter was identified in erythrocyte membranes by the immunoblotting technique, using both antibodies raised against purified glucose transporter and anti-idiotypic antibodies. Two-dimensional peptide mapping revealed substantial peptide homology between glucose transporter and senescent cell antigen. In addition, the glucose transporter shared peptide homology with band 3 and its defined proteolytic fragments located toward the carboxyl terminus of band 3. Peptide homology between glucose transporter and the Mr approximately equal to 41,000 cytoplasmic segment of band 3 that contains the amino terminus could not be demonstrated. Thus, glucose transporter appears to be part of or derived from band 3, and to share substantial peptide homology with senescent cell antigen.     

Lentropin, a protein that controls lens fiber formation, is related functionally and immunologically to the insulin-like growth factors.

Lentropin, a factor present in the vitreous humor of the eye, stimulates lens fiber differentiation from chicken embryo lens epithelial cells in vitro. Lentropin has been partially purified but has not been isolated in sufficient quantity or purity for direct comparison with other growth and differentiation factors. Previous studies have shown that insulin and fetal bovine serum share with lentropin the ability to stimulate lens fiber formation from cultured epithelial cells. In the present study, a number of hormones and growth factors were assayed for lentropin activity. Of those tested, the only substances that had this activity were the insulin-like growth factors (IGFs) somatomedin C (Sm-C/IGF-I) and multiplication-stimulating activity (MSA/IGF-II). Sm-C/IGF-I was approximately 30 times more potent than insulin or MSA/IGF-II in promoting fiber cell formation. A monoclonal antibody to human Sm-C/IGF-I inhibited purified Sm-C/IGF-I, fetal bovine serum, and chicken vitreous humor from stimulating fiber cell differentiation in vitro. This antibody has been shown not to crossreact with insulin and did not block insulin-stimulated lens fiber formation. These findings indicate that lentropin is related to the IGFs and that these factors may play important roles in controlling cell differentiation, in addition to their better-known ability to stimulate cell division.     

Evidence for a novel DNA damage binding protein in human cells.

We describe a novel DNA damage binding activity in nuclear extracts from a normal human fibroblast cell strain. This protein was identified using electrophoretic mobility shift assays of immunopurified UV-irradiated oligonucleotide substrates containing a single, site-specific cyclobutane pyrimidine dimer or a pyrimidine (6-4) pyrimidinone photoproduct. Compared with the (6-4) photoproduct, which displayed similar levels of binding in double and single-stranded substrates, the protein showed somewhat lower affinity for the cyclobutane dimer in a single-stranded oligonucleotide and negligible binding in double-stranded DNA. The specificity and magnitude of binding was similar in cells with normal excision repair (GM637) and repair-deficient cells from xeroderma pigmentosum groups A (XP12RO) and E (XP2RO). An apparent molecular mass of 66 kDa consisting of two subunits of approximately 22 and approximately 44 kDa was determined by Southwestern analysis. Cell cycle studies using centrifugal cell elutriation indicated that the binding activity was significantly greater in G1 phase compared with S phase in a human lymphoblast cell line. Gel supershift analysis using an anti-replication protein A antibody showed that the binding protein was not antigenically related to the human single-stranded binding protein. Taken together, these data suggest that this activity represents a novel DNA damage binding protein that, in addition to a putative role in excision repair, may also function in cell cycle or gene regulation.     

Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor.

A human cDNA coding for a protein related to the vascular permeability factor (VPF) was isolated from a term placenta cDNA library; we therefore named its product placenta growth factor (PlGF). PlGF is a 149-amino-acid-long protein and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human VPF. Computer analyses reveal a putative signal peptide and two probable N-glycosylation sites in the PlGF protein, one of which is also conserved in human VPF. By using N-glycosidase F, tunicamycin, and specific antibodies produced in both chicken and rabbit, we demonstrate that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium. In addition, PlGF, like VPF, proves to be a dimeric protein. Finally, a conditioned medium from COS-1 cells containing PlGF is capable of stimulating specifically the growth of CPA, a line of endothelial cells, in vitro.     

Cultured human umbilical vein endothelial cells contain a membrane glycoprotein immunologically related to platelet glycoprotein Ib

Using a platelet glycoprotein Ib (GpIb)-specific monoclonal antibody, AP-1, we have studied cultured human umbilical vein endothelial cells (HUVEC) for the presence of GpIb. Radiolabeled AP-1 bound specifically and saturably to HUVEC in suspension and detected a single class of binding sites (100,000/cell). When Triton X-100 extracts of HUVEC were chromatographed on wheat germ agglutinin (WGA)-Sepharose, radioiodinated, precipitated with AP-1, and subjected to reduced sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), major radioactive bands of 228,000, 145,000, and 130,000 were seen. The latter two bands correspond to the 156,000 and 140,000 bands, representing GpIb alpha and glycocalicin, respectively, which are seen when platelets are subjected to the same procedure. The 228,000 band corresponds to a band previously noted in immunoprecipitates of platelet GpIb but not fully explained. When HUVEC were grown in the presence of 35S-methionine, extracted with Triton X-100, chromatographed on WGA-Sepharose, immunoprecipitated with AP-1, and subjected to reduced SDS-PAGE, radioactive bands of 210,000, 156,000, and 90,000 were seen. We conclude that cultured HUVEC synthesize and express on their surface a glycoprotein immunologically related to platelet GpIb.     

Production of a factor by cultured human heart valves that is immunologically related to interleukin 1

Analysis of the conditioned medium from cultured human heart valves showed that these tissues secrete a biologically active factor that induces chondrocytes in cultured cartilage to degrade extracellular matrix proteoglycan. This activity was similar to that described for porcine interleukin-1 (catabolin) and a cytokine secreted by cultured porcine heart valves (cardiac catabolic factor). The biological activity of the material in human valve conditioned medium was unaffected by the presence of low doses of cortisol, but its production by cultured valves was impaired by this steroid or benoxaprofen and abolished by cycloheximide. Addition of the conditioned medium to fibroblast monolayers stimulated the secretion of prostaglandin E and the tissue inhibitor of metalloproteinases (TIMP) but not collagenase. Preincubation of the conditioned medium with antiserum raised to the acidic form of porcine interleukin-1 neutralised the proteoglycan degrading stimulus. The material is biologically similar to other cytokines and antigenically related to porcine interleukin-1.     

Evidence for the presence of lipofibroblasts in human lung

The lipid-containing alveolar interstitial fibroblast (lipofibroblast) is known to be critically involved in rodent lung development, homeostasis, and injury/repair. However, there is lack of information on their presence and function in the human lung. Based on a number of morphological (lipid staining), molecular (presence of characteristic lipogenic and absence of myogenic markers), and functional (triglyceride uptake) characteristics that are the hallmarks of the rodent lung lipofibroblast, using human lung fibroblasts of embryonic (WI-38) and adult origin and lung tissue from human autopsy specimens, the authors for the first time clearly demonstrate the presence of lipofibroblasts in the human lung.     

Platelets express a membrane protein complex immunologically related to the fibroblast fibronectin receptor and distinct from GPIIb/IIIa

We have previously identified and characterized a membrane glycoprotein complex (GP150/135) that functions as fibronectin receptor (FN-R) in fibroblast adhesion. Here we report that an immunologically related protein complex is expressed at the surface of human platelets. Antibodies monospecific for the smaller subunit (GP135) of the fibroblast FN-R in fact specifically stained the platelet surface, as determined by FACS analysis, and reacted with a component of molecular weight (mol wt) 138,000 as shown in western blots of platelet membranes. Moreover, the same antibodies precipitated the 138,000 component together with a 160,000 protein, suggesting that the two molecules are associated in a supramolecular complex. A comparative analysis indicated that this protein complex is distinct from the GPIIb/IIIa complex, known to function as a receptor of wide specificity for fibrinogen, fibronectin, and von Willebrand factor. Differential extraction experiments revealed that the platelet 138,000 component is an integral membrane protein.     

Characterization of a protein, released by the T47D cell line, immunologically related to the major envelope protein of mouse mammary tumor virus.

The T47D human mammary adenocarcinoma cell line in vitro releases viral particles as well as soluble proteins. Both fractions were shown to contain antigens that immunologically crossreact with the major glycoprotein (gp52) of mouse mammary tumor virus. The crossreacting antigens are located on polypeptides with apparent molecular weights of about 68,000 and 60,000. The larger one is present in viral particles whereas both are found in the soluble fraction. Both proteins are glycosylated. The human tissue culture proteins differ from gp52 not only in molecular weight but also in charge heterogenity and in polypeptide profiles obtained after partial proteolysis. The results suggest that there is a restricted similarity between MMTV gp52 and the immunologically related T47D proteins.     

Polypeptides immunologically related to band 3 are present in nucleated somatic cells.

Band 3, the major transmembrane polypeptide of erythrocytes, mediates the exchange of anions (chloride and bicarbonate) across the membrane. We suspected that band 3 was present on nucleated somatic cells as well as erythrocytes because the senescent cell antigen that is immunologically related to band 3 is present on lymphocytes, platelets, adult liver cells, and embryonic kidney cells; and antibodies prepared against the senescent cell antigen isolated from leukocytes react with erythrocyte band 3. For this reason, we examined human fibroblasts, lung cells, neutrophils, mononuclear leukocytes, squamous epithelial (mouth) cells, lung squamous epithelial carcinoma, mouse neuroblastoma cells, and rat hepatocytes for immunoreactive forms of band 3 by using monospecific antibodies to erythrocyte band 3. The results demonstrated that polypeptides sharing common antigenic determinants with erythrocyte band 3 are present in nucleated somatic cells as determined by immunofluorescence, immunoelectron microscopy, and immunoautoradiography. Peptide mapping revealed substantial sequence homology between erythrocyte band 3 and the band 3-like protein of leukocytes. Immunofluorescence studies indicate that the band 3-like proteins in nucleated cells participate in antibody-induced cell surface capping.     

Identification and cloning of a secreted protein related to the cysteine-rich domain of frizzled. Evidence for a role in endothelial cell growth control.

We report the isolation of a cDNA, FrzA (frizzled in aorta; GenBank accession No. U85945), from bovine aortic endothelium. It is the bovine counterpart of the mouse sFRP1, which encodes for a secreted protein that is homologous to the cysteine-rich domain of frizzled. Members of the frizzled family of genes have been shown to be required for tissue polarity and to act as receptors for Wnt. The predicted protein product of this gene includes the cysteine-rich extracellular domain, but not the 7 putative transmembrane domains that are highly conserved among members of the frizzled family. Visualization of FrzA mRNA and protein revealed that it was widely distributed among adult tissues. FrzA is expressed by highly differentiated or polarized cells, eg, neurons, cardiocytes, or various epithelia. Analysis of its expression in endothelium revealed that FrzA mRNA levels were high in endothelial cells scraped from freshly obtained bovine aortas, decreased when cells were placed in culture and began to proliferate, but increased at confluence. Transient transfection assays and an assay using addition of purified protein indicate that FrzA reduces the proliferation of endothelial cells. These data demonstrate the existence of a secreted protein homologous to the extracellular domain of the fz receptor, which we speculate plays a role in controlling cell growth and differentiation, possibly by regulating accessibility to Wnt family members.     

Detection in human breast carcinomas of an antigen immunologically related to a group-specific antigen of mouse mammary tumor virus

An antigen immunologically related to a group-specific antigen (gp52, a 52,000-dalton glycoprotein) of the mouse mammary tumor virus has been identified in paraffin sections of human breast cancers by means of the indirect immunoperoxidase technique. The specificity of the reaction with antibody against mouse mammary tumor virus was examined by absorption of the IgG with the following: (a) purified gp52; (b) a number of virus preparations (mouse mammary tumor virus, Rauscher leukemia virus, simian sarcoma virus, baboon endogenous virus, and Mason-Pfizer monkey virus); (c) normal plasma, leukocytes, breast tissue, milk, actin, collagen, and hyaluronic acid, all of human origin; (d) sheep erythrocytes and mucin. Only mouse mammary tumor virus (from C(3)H or Paris RIII strains and grown in either murine or feline cells) and purified gp52 eliminated the immunohistochemical reaction in the human breast tumors.Positive reactions were seen in 51 of 131 (39%) breast carcinomas of various histologic types, a minimal estimate in view of the limited number of sections from each tumor that could be examined. Negative reactions were obtained in all 119 benign breast lesions (cystic disease, fibroadenoma, papilloma, gynecomastia) and in all 18 normal breast tissues. With one exception, 99 carcinomas from 13 organs other than breast and 8 cystosarcomas were all negative.     

Characterization of cDNA encoding a human sperm membrane protein related to A4 amyloid protein.

A rat testis lambda gt11 cDNA library was screened with a monoclonal antibody raised against a human sperm membrane protein designated YWK-II. A clone was found with a cDNA insert composed of 1837 base pairs that contained an open reading frame coding for 191 amino acid residues. The deduced polypeptide contained a segment with high homology to the transmembrane-cytoplasmic domains of the A4 amyloid protein found in brain plaques of Alzheimer disease patients. A sequence of basic amino acid residues, Arg-Lys-Arg, was found instead of Lys-Lys-Lys at the probable membrane-cytoplasmic junction that may be a unique property of sperm membrane proteins.     

Evidence that the 60-kDa protein of 17S U2 small nuclear ribonucleoprotein is immunologically and functionally related to the yeast PRP9 splicing factor and is required for the efficient formation of prespliceosomes.

Small nuclear ribonucleoprotein (snRNP) U2 functions in the splicing of mRNA by recognizing the branch site of unspliced mRNA. The binding of U2 snRNP and other components to pre-mRNA leads to the formation of a stable prespliceosome. In HeLa nuclear extracts, U2 snRNP exists either as a 17S form (under low salt conditions) or a 12S form (at higher salt concentrations). We have recently shown that the purified 17S U2 snRNP contains nine proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa in addition to the common snRNP proteins and the U2 proteins A' and B" that are found in the 12S U2 snRNP form. By using antibodies against the PRP9 protein from Saccharomyces cerevisiae (a protein required for the addition of U2 to prespliceosomes in yeast), we have shown that the 60-kDa protein specific to human U2 snRNP particles is structurally related to the yeast PRP9 protein. Interestingly, anti-PRP9 antibodies strongly inhibit prespliceosome formation in HeLa nuclear splicing extracts, resulting in a complete inhibition of the mRNA splicing reaction in vitro. This indicates that the U2 60-kDa protein may also be functionally related to its yeast counterpart PRP9. Most importantly, the addition of purified 17S U2 snRNPs, but not of 12S U2 snRNPs, to HeLa splicing extracts in which the endogeneous U2 snRNPs have been functionally neutralized with anti-PRP9 antibodies fully restores the mRNA-splicing activity of the extracts. These data suggest further that the 17S form is the functionally active form of U2 snRNP in the spliceosome.