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G R Nemerow

Publications and source records attributed to G R Nemerow.

At least 19 recordsLinked to original sources
PubMed1991

Inhibition of Epstein-Barr virus infection in vitro and in vivo by soluble CR2 (CD21) containing two short consensus repeats.

The extracellular domain of CR2, the Epstein-Barr virus (EBV)/C3d receptor of B lymphocytes, contains 15 or 16 tandemly arranged short consensus repeat elements (SCR). Recombinant CR2 proteins containing SCR 1 and 2 fused to Staphylococcus aureus protein A (PA-CR2) and to murine complement factor H SCR 20 (CR2FH) were expressed in Escherichia coli and in insect cells, respectively. These recombinant CR2 molecules retained functional activity as indicated by their ability to bind to C3dg in an enzyme-linked immunosorbent assay and to inhibit EBV gp350/220 binding to B cells. PA-CR2 and CR2FH were as efficient in blocking EBV gp350/220 binding as the full-length CR2 extracellular domain, indicating that the first two SCR of CR2 contain the majority of the ligand binding activity of the receptor. PA-CR2 and CR2FH inhibited EBV-induced B-cell proliferation in vitro and blocked the development of EBV-induced lymphoproliferative disease in severe combined immunodeficient mice reconstituted with human lymphocytes. These studies indicate that soluble forms of truncated CR2 proteins may have potential therapeutic value in the treatment of EBV-induced lymphoproliferative disorders in humans that involve viral replication.

Amino Acid Sequence↗
PubMed1990

CR2 complement receptor.

CR2, a membrane glycoprotein, is one of a number of cell-surface proteins which bind activation and processing fragments of the complement system. CR2, which is found on normal B lymphocytes, follicular dendritic cells in lymphoid organs, and epithelial cells, interacts preferentially with C3dg, the terminal activation/processing fragment of the third complement component. Attachment of C3dg to CR2 brings complement activators, bearing covalently bound C3dg, into direct membrane contact with CR2-bearing cells. Epstein-Barr virus, a human herpesvirus, also binds to CR2 on B lymphocytes. Attachment of EBV is followed by infection. CR2 has been purified and the binding properties of its ligands analyzed. Monoclonal antibodies have been developed and used to probe the structural correlates of CR2 functions. CR2 has been molecularly cloned and its primary amino acid sequence deduced. These data indicate that it shares characteristic structural features with a number of other complement and non-complement cell membrane and plasma proteins. Several of the complement-associated proteins in this family possess regulatory functions; they are encoded by linked genes which have been localized to band q32 on chromosome 1. CR2 has been expressed in primate and rodent cells by transfection of cDNA in antigenically and functionally intact form. It has also been expressed in soluble form and its structure, electron microscopic appearance and binding characteristics analyzed in detail. The present state of knowledge of the structure and genetics of CR2 and current understanding of its biologic functions are summarized here.

Animals↗
PubMed1990

Soluble recombinant CR2 (CD21) inhibits Epstein-Barr virus infection.

Epstein-Barr virus (EBV), an oncogenic herpesvirus of humans, displays selective tropism for B lymphocytes and epithelial cells. EBV tropism is thought to be determined in part by a unique host cell receptor termed CR2 (CD21). Although previous studies have demonstrated that CR2 mediates EBV binding to B cells, its role in initiating EBV infection and B-cell transformation is less certain. In the studies reported here, soluble recombinant CR2 was shown to cause substantial inhibition of EBV infection of B cells in vitro, indicating that CR2 binding initiates EBV infection. Soluble CR2 may represent a therapeutic agent for acute and chronic EBV infections in humans.

Antigens, CD↗
PubMed1989

Hydrodynamic, electron microscopic, and ligand-binding analysis of the Epstein-Barr virus/C3dg receptor (CR2).

The interaction of the Epstein-Barr virus/45-kDa proteolytic fragment of C3 (C3dg) receptor (CR2) with its viral ligand, the Epstein-Barr virus glycoprotein gp350/220, initiates the sequence of events leading to virus internalization and B lymphocyte transformation. Soluble recombinant receptor (rCR2) and gp350/220 as well as the natural ligand, C3dg, were subjected to a number of analytical techniques including gel permeation chromatography, density gradient ultracentrifugation, circular dichroism, and electron microscopy in order to determine their hydrodynamic, structural, and binding properties. Both rCR2 and gp350/220 were found to be highly extended proteins (f/fo = 2.1 and 2.4/2.2, respectively). C3dg, in contrast to the viral ligand, is only somewhat elongated (f/fo = 1.5). Soluble rCR2, visualized by high resolution electron microscopy, was shown to be an extended, highly flexible molecule comprised of ringlet domains, each approximately 24.1 A in length, which likely correspond to the short consensus repeat motif deduced from the CR2 cDNA nucleotide sequence. Ligand-binding studies carried out under physiological conditions indicated that gp350/220 binding to rCR2 was saturable and univalent, with a dissociation constant of 3.2 nM. In contrast, monomeric C3dg did not bind to rCR2 under physiological conditions; however, at reduced ionic strength, monomeric C3dg binding could be measured. These studies indicate that the affinity of the C3dg monomer for rCR2 under physiologic conditions is approximately 10(4)-fold less than that of the viral ligand. The molecular properties of rCR2 revealed in these studies provide essential information for future studies of the biologic functions of the Epstein-Barr virus/C3dg receptor.

Antigens, CD↗
PubMed1989

Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2).

The Epstein-Barr virus gp350/220 envelope protein mediates virus attachment to the EBV/C3dg receptor (CR2) of human B lymphocytes. Synthetic peptides corresponding to two regions in gp350/220, which have a similar amino acid sequence with the complement C3dg protein, were used to identify a receptor binding epitope. A peptide corresponding to the N terminus of gp350/220, EDPGFFNVE, bound to purified CR2 and to CR2 positive but not CR2 negative B and T lymphoblastoid cell lines. Soluble monomeric gp350/220 peptide blocked CR2 binding to immobilized EBV, while multimeric forms of the N-terminal gp350/220 peptide conjugated to albumin efficiently blocked recombinant gp350/220 and C3dg binding to B cells as well as EBV-induced B cell proliferation and transformation. These studies indicate that the N-terminal region of gp350/220 plays a crucial role in mediating the earliest stages of EBV infection of B cells and provides a molecular basis for the restricted host cell EBV tropism.

Amino Acid Sequence↗
PubMed1989

Complement and infectious agents: a tale of disguise and deception.

The primary functions of the immune system center on the recognition and elimination of infectious agents, foreign substances, and altered or transformed host cells. Bacterial and viral infections represent major challenges to the immune system because of the ability of these agents to replicate and injure host cells and tissues, and to impair recognition and elude destruction by humoral or cellular defense mechanisms. Complement functions at multiple levels in the control of bacterial infections: recognition, in vivo clearance, opsonization, killing and lytic destruction. Although bacteria frequently evade destruction and cause disease, the ultimate control of bacterial infections is largely dependent on the actions of antibody and complement functioning synergistically with phagocytic cells. The crucial role of complement in these actions is apparent in the striking susceptibility to life-threatening bacterial infections of most individuals who are genetically deficient in complement components, particularly C3, C3 regulatory proteins and C5, C6, C7 or C8. The complement system also interacts with viruses and virus-infected cells at multiple levels and, as in the case of bacteria, viruses have evolved various mechanisms to avoid destruction. As obligate intracellular parasites which reside in host cells; however, the elimination of viruses is largely dependent on cellular immune responses. Complement also interacts with parasites and fungi; however, the physiologic importance of these reactions has yet to be elucidated. The various types of interactions of infectious agents with the complement system and the mechanisms which these pathogens have evolved to evade destruction and, in some cases, to potentiate and facilitate infection are summarized here.

Bacterial Infections↗
PubMed1988

Epstein-Barr virus regulates activation and processing of the third component of complement.

Serum incubated with purified EBV was found to contain C3 cleavage fragments characteristic of C3c. Since the cofactors necessary for such cleavage of C3b by factor I are not normally present in serum, EBV was tested for factor I cofactor activity. Purified EBV from both human and marmoset EBV-producing cell lines was found to act as a cofactor for the factor I-mediated breakdown C3b to iC3b and iC3b to C3c and C3dg. EBV also acted as a cofactor for the factor I-mediated cleavage of C4b to iC4b and iC4b to C4c and C4d. EBV from both the human and marmoset cell lines accelerated the decay of the alternative pathway C3 convertase. The classical pathway C3 convertase was unaffected. Multiple lines of evidence eliminated the possibility that marmoset or human CR1 was responsible for the functional activities of EBV preparations. The spectrum of activities was different from CR1 in that EBV and EBV-expressing cell lines failed to rosette with C3b or particles bearing C3b, the primary functional assay for CR1, and EBV did not accelerate classical pathway C3 convertase decay, another property of CR1. In addition, CR1 could not be detected immunologically on marmoset or human EBV-expressing cells and mAbs to CR1 failed to alter EBV-produced decay acceleration and factor I cofactor activities, although the antibodies blocked the same CR1-dependent functional activities. The multiple complement regulatory activities exhibited by purified EBV derived from human and marmoset cells differ from those of any of the known C3 or C4 regulatory proteins. These various activities would be anticipated to provide survival value for the virus by subverting complement- and cell-dependent host defense mechanisms.

Animals↗
PubMed1988

Activation of the alternative complement pathway by EBV and the viral envelope glycoprotein, gp350.

The EBV-producing B lymphoblastoid cell line B95-8 was found to efficiently activate the alternative C pathway whether assessed with Mg-EGTA-treated human serum or with mixtures of the purified proteins of the pathway (PAP). The ability of the cells to activate was markedly increased after stimulation of EBV replication by treatment of the cells with a phorbol ester, and decreased by treatment of the cells with a viral polymerase inhibitor. Alternative pathway activation was dependent on the presence of either properdin or EBV-immune IgG; the addition of either alone to the PAP led to the deposition of 200,000 C3 molecules/cell. The addition of both properdin and immune IgG to the PAP markedly increased C3 binding to a level of 800,000 molecules/cell. Several lines of evidence indicate that the major external glycoprotein of EBV, gp350, mediates alternative pathway activation by B95-8 cells. First, the ability to activate C positively correlated with gp350 expression on the surface of the EBV-producing cells and gp350- cells failed to activate; second, the anti-EBV antibody in immune human sera which enhanced activation specifically immunoprecipitated gp350 from membranes of B95-8 cells; third, a significant proportion of the C3 which became bound to the cells during activation was attached either to gp350 or to the anti-gp350 antibody found in immune human sera; and fourth, purified gp350, as well as EBV, efficiently activated the alternative pathway. These results indicate that gp350, an EBV envelope glycoprotein, is an efficient alternative pathway activator and its expression on cell membranes is associated with the ability to activate C.

Animals↗
PubMed1988

CR2 is a complement activator and the covalent binding site for C3 during alternative pathway activation by Raji cells.

Antibody-independent activation of the alternative C pathway by human lymphoblastoid cell lines latently infected with EBV has been recognized for some time, although the mechanisms involved and the specific cell surface molecule(s) recognized by the C system have not been identified. The present studies, carried out with the purified proteins of the alternative pathway have addressed these questions. Activation of the purified proteins of the alternative pathway by Raji lymphoblastoid cells was found to be antibody independent, confirming earlier findings with serum. Surprisingly, activation was highly dependent on properdin. In other models properdin has been found to augment alternative pathway activation and to be required for lysis of virus infected cells. Molecules which activate the alternative pathway provide binding sites on which C3 breakdown by regulatory proteins is impeded; therefore intact C3b accumulates on the activator. Immunoprecipitation studies with either anti-CR2 or anti-C3 have identified CR2, the R for C3d,g and EBV, as a major covalent and noncovalent binding site for C3 deposition on Raji cells during alternative pathway activation. Covalently bound C3b was dissociated from CR2 by hydroxylamine, indicating attachment via an ester bond. C3b binding after activation was not reduced by an anti-CR2 mAb which blocks CR2 R function, indicating that it was probably not mediated by C3d,g R epitopes on CR2. Direct confirmation of the ability of CR2 to trigger the alternative pathway came from studies with purified CR2 which was found to activate the alternative C pathway in serum or in mixtures of the purified proteins of the pathway. This work provides conclusive evidence that CR2 is a C activator and functions in this capacity on Raji cells.

Burkitt Lymphoma↗
PubMed1987

Virus receptors on lymphoid cells.

The studies described above indicate the advances made in the isolation and characterization of virus receptors of lymphoreticular cells (Table I). Although the examples of lymphotropic virus receptors cited in this chapter indicate that single membrane glycoproteins can serve as receptors, other nonlymphoid viruses such as vesicular stomatitis virus (VSV) (Table I) appear to utilize glycolipid or phospholipid components for cell attachment. These molecules may be responsible for the broad specificity of host cell attachment by these viruses. The virus-binding moiety of phospholipid/glycolipid receptors remains to be fully analyzed. It is anticipated that biochemical techniques such as the use of chemical cross-linking reagents will aid in the identification of other virus receptors such as CMV and measles which have less restricted lymphotropism than EBV. In addition, X-ray crystallographic analysis of viruses such as the recent studies of human rhinovirus and poliovirus may provide insights on the complementary structure of cellular recognition sites for viruses.

B-Lymphocytes↗
PubMed1987

Molecular cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes.

Complementary DNA clones for complement receptor type 2 (CR2), the B-lymphocyte membrane protein that serves as the receptor for Epstein-Barr virus and the C3d complement fragment, were obtained by screening a lambda gt11 library generated from Raji B lymphoblastoid cell mRNA. A 4.2-kilobase (kb) clone, representing the entire coding sequence of the protein plus untranslated 5' and 3' nucleotide sequences was obtained and sequenced. The 4.2-kb clone, which contains all but about 500 base pairs (bp) of the 5' untranslated region of the full-length CR2 mRNA, consists of 63 bp of 5' untranslated nucleotide sequence followed successively by a start codon, a 20-amino acid hydrophobic signal peptide, 1005 amino acids having a repeating motif, a 28-amino acid probable transmembrane domain, and a 34-amino acid cytoplasmic tail. The deduced amino acid sequence of the protein indicates that the extracellular domain consists entirely of 16 tandemly arranged repeating elements, each 60-75 amino acids in length, which are identified by multiple conserved residues. This repeating motif also occurs in the C3b/C4b receptor, several complement proteins, and a number of noncomplement proteins. In CR2, the 16 repeats occur in four clusters of four repeats each. Approximately 10% of the deduced amino acid sequence, including the amino and carboxyl termini, was confirmed by amino acid sequencing of tryptic peptides derived from purified CR2. The nucleotide and derived amino acid sequence of CR2 and related studies are presented here.

Amino Acid Sequence↗
PubMed1987

Identification of gp350 as the viral glycoprotein mediating attachment of Epstein-Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d.

The major Epstein-Barr virus (EBV) envelope glycoprotein, gp350, was purified from the B95-8 cell line and analyzed for its ability to mediate virus attachment to the isolated EBV/C3d receptor (CR2) of human B lymphocytes. Purified gp350 and EBV, but not cytomegalovirus, exhibited dose-dependent binding to purified CR2 in dot blot immunoassays. Binding was inhibited by certain monoclonal antibodies to CR2 and to gp350. Liposomes bearing incorporated gp350 bound to CR2-positive B-cell lines but not to CR2-negative lines. Liposome binding was also inhibited by the OKB7 anti-CR2 monoclonal antibody. A computer-generated comparison of the deduced gp350 amino acid sequence with that of the human C3d complement fragment revealed two regions of significant primary sequence homology, a finding which suggests that a common region on these two unrelated proteins may be involved in CR2 binding.

Amino Acid Sequence↗
PubMed1986

Incorporation of the purified Epstein Barr virus/C3d receptor (CR2) into liposomes and demonstration of its dual ligand binding functions.

The 145-kDa molecule that has been identified as the C3d receptor CR2 was isolated from lysates of Raji cells by affinity chromatography by using the monoclonal antibody (MoAb)HB-5. The purified protein was incorporated into 14C-phosphatidylcholine liposomes by deoxycholate dialysis followed by flotation on discontinuous sucrose gradients. Incorporation of the receptor was verified by testing the gradient fractions for CR2 by an enzyme-linked immunosorbent assay. Liposomes were shown to be unilamellar vesicles ranging in diameter from 25 to 100 nm by electron microscopy. The external orientation of CR2 in the membranes was demonstrated by immunoelectron microscopy. The functional activities of liposomes containing CR2 and liposomes without protein were compared. CR2 liposomes bound to EC3d, but not to E, and this binding was inhibited by the anti-CR2 MoAb OKB7 and by a MoAb specific for C3d. Control liposomes failed to bind to either E or EC3d. The ability of CR2 to function as a receptor for Epstein Barr virus (EBV) was tested in two ways. First, CR2 liposomes bound to B95-8, a cell line expressing EBV membrane antigens, but not to B95-8 cells treated with the viral DNA polymerase inhibitor phosphonoformic acid. Second, liposomes containing CR2 were shown by ultracentrifugal analyses to bind directly to purified EBV, and this binding was also inhibited by OKB7. Control liposomes did not bind to B95-8 cells or to EBV. These findings show that CR2 purified from detergent extracts of Raji cells can be reconstituted into lipid membranes with maintenance of its dual functions as a receptor for C3d and EBV.

Animals↗
PubMed1986

Biochemical and antigenic analysis of the Epstein Barr virus/C3d receptor (CR2).

Four monoclonal antibodies (OKB7, HB-5, AB-1, and anti-B2) that recognize a 145-kDa B cell-specific membrane structure have markedly different abilities to 1) inhibit C3d and EBV binding to B cells, 2) immunoprecipitate a 145-kDa B cell protein, and 3) stimulate B cell proliferation and differentiation into Ig-secreting cells. This study was initiated to determine whether these four monoclonal antibodies (MoAb) react with the same protein; a related goal was to determine whether the structure(s) recognized by these antibodies constitutes an antigenically related family of structurally distinct molecules. In the studies presented here, the four MoAb were found to fully immunoprecipitate the purified 145-kDa B cell molecule isolated by immunoaffinity chromatography on either OKB7, HB-5, or AB-1 columns, findings that show conclusively that the antibodies all react with the same B cell protein. The variable ability to immunoprecipitate this B cell membrane protein was found to result from differences in exposure or accessibility of the relevant antigenic epitopes in the detergent extract. The 145-kDa molecule immunoprecipitated with the four MoAb was equivalently sensitive to endoglycosidase F and yielded the same banding pattern after digestion with endoglycosidase F and after partial digestion with either S. aureus V8 protease or with trypsin. Within the limits of the sensitivity of these techniques, therefore, there is no evidence for carbohydrate or protein differences in the EBV/C3d receptor (CR2) molecule recognized by the four MoAb. Additional studies showed that the four MoAb react with distinct and nonoverlapping antigenic epitopes on the 145-kDa molecule. The variable abilities of the four MoAb to inhibit CR2 function and EBV binding and to trigger B cell activation, together with the other findings noted above, indicates that the 145-kDa EBV/C3d receptor possesses discretely localized functional domains.

Antibodies, Monoclonal↗
PubMed1986

Purification of the Epstein-Barr virus/C3d complement receptor of human B lymphocytes: antigenic and functional properties of the purified protein.

The Epstein-Barr virus/C3d receptor (CR2) of human B lymphocytes was purified to homogeneity from Raji cells by immunoaffinity chromatography. The average yield of the 145-kilodalton receptor was 400 pmol (50 micrograms) per 10(10) cells, representing an approximate 75% recovery. The isolated 145-kilodalton protein was antigenically and functionally intact as it reacted with several anti-CR2 monoclonal antibodies and bound purified Epstein-Barr virus and C3d,g. These findings with the purified molecule provide an unequivocal demonstration of the dual receptor functions of this protein.

B-Lymphocytes↗
PubMed1985

Complement effector mechanisms in health and disease.

Complement is an effector system able to mediate a number of biological activities in vitro and in vivo. Most familiar is the ability of the system to mediate the lytic destruction of numerous kinds of cells and pathogenic organisms including bacteria, viruses, and virus-infected cells. In addition, the complement system also activates neutrophils, monocytes, basophils, mast cells, and lymphocytes to perform specialized functions. While generally considered to be confined to the effector side of immune reactions, recent evidence indicates that the complement system also directly recognizes and is triggered by a number of bacteria and viruses as well as virus-infected cells in the absence of antibody. In such reactions, complement fulfills the recognition role normally associated with the antibody molecule or immune lymphocyte. The complement system may thus also function as a natural surveillance system operative prior to the induction of specific immunity. Involvement of the complement system in biological reactions has been ascertained by several techniques over the years. These include quantitation of individual complement components in human sera and demonstration of complement deposition in diseased tissues in human diseases and in experimental diseases in animals. Such techniques, however, have limitations in specificity and sensitivity. Assays which detect specific features of the complement activation process have become available in recent years. These tests detect the physical, chemical, or antigenic changes characteristic of the complement activation process. These assays are extremely specific and quantitative; furthermore, most are usable with samples from patients. Three general approaches have been utilized to develop such specific quantitative assays for complement activation. The first includes assays which quantitate activation-specific limited proteolysis of the complement components. The second type of assay includes tests which detect and quantitate new antigens or other activation-specific antigenic changes. The third category is represented by assays which detect and quantitate the protein-protein complexes characteristic of the activation process. Examples of tests presenting each of these approaches are given.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗