Probing the Origin of the Toxicity of Oligomeric Aggregates of α-Synuclein with Antibodies.

The aggregation of α-synuclein, a protein involved in neurotransmitter release at presynaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions, most notably Parkinson's disease. Intraneuronal inclusion bodies, primarily composed of α-synuclein fibrils, are the major histopathological hallmarks of these disorders, although small oligomeric assemblies are believed to play a crucial role in neuronal impairment. We have probed the mechanism of neurotoxicity of α-synuclein oligomers isolated in vitro using antibodies targeting the N-terminal region of the protein and found that the presence of the antibody resulted in a substantial reduction of the damage induced by the aggregates when incubated with primary cortical neurons and neuroblastoma cells. We observed a similar behavior in vivo using a strain of C. elegans overexpressing α-synuclein, where the aggregation process itself is also partially inhibited as a result of incubation with the antibodies. The similar effects of the antibodies in reducing the toxicity of the aggregated species formed in vitro and in vivo provide evidence for a common origin of cellular impairment induced by α-synuclein aggregates.

Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography.

Nucleophosmin (NPM1) is a multifunctional phosphoprotein which plays important roles in diverse biological processes. NPM1 can form homo- or hetero-oligomers through its N-terminal region, and bind DNA and RNA through its C-terminal region. However, the monomer-oligomer distribution of NPM1, and the extent of NPM1 binding and unbinding to RNA in living cells, are not fully understood. In this work, we analysed molecular complexes of NPM1 using size exclusion chromatography. We found that a substantial fraction of NPM1 behaves as an oligomer in HeLa cells. Furthermore, we identified three distinct oligomeric states of NPM1 using molecular characterization techniques such as subcellular localization and RNA binding. Finally, we found that heterozygous expression of a leukemia-associated NPM1 mutant significantly decreases the RNA binding level. Our data demonstrate that size exclusion chromatography provides a powerful tool for analysing NPM1 oligomers.

In planta production of ELPylated spidroin-based proteins results in non-cytotoxic biopolymers.

BACKGROUND: Spider silk is a tear-resistant and elastic biopolymer that has outstanding mechanical properties. Additionally, exiguous immunogenicity is anticipated for spider silks. Therefore, spider silk represents a potential ideal biomaterial for medical applications. All known spider silk proteins, so-called spidroins, reveal a composite nature of silk-specific units, allowing the recombinant production of individual and combined segments. RESULTS: In this report, a miniaturized spidroin gene, named VSO1 that contains repetitive motifs of MaSp1 has been synthesized and combined to form multimers of distinct lengths, which were heterologously expressed as elastin-like peptide (ELP) fusion proteins in tobacco. The elastic penetration moduli of layered proteins were analyzed for different spidroin-based biopolymers. Moreover, we present the first immunological analysis of synthetic spidroin-based biopolymers. Characterization of the binding behavior of the sera after immunization by competitive ELISA suggested that the humoral immune response is mainly directed against the fusion partner ELP. In addition, cytocompatibility studies with murine embryonic fibroblasts indicated that recombinant spidroin-based biopolymers, in solution or as coated proteins, are well tolerated. CONCLUSION: The results show that spidroin-based biopolymers can induce humoral immune responses that are dependent on the fusion partner and the overall protein structure. Furthermore, cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that recombinant produced biopolymers composed of spider silk-like repetitive elements are suitable for biomedical applications.

[Glycopolymers of microorganisms: achievements and future research (review)].

This review presents the current literature data on the structure of peptidoglycans, lipopolysaccharides, teichoic acids, the mechanism of biological action of lipopolysaccharides, and the possibility of uising oligosaccharides for creation of glycoconjugate vaccines, as well as promising areas for further research of glycopolymers of microorganisms.

Análisis de las polihidroxialcanoato sintasas (PhaC1 y PhaC2) en una cepa de Pseudomonas fluorescens IBUN S1602, aislada en suelos colombianos / Analysis of polyhydroxyalkanoate synthases (PhaC1 and PhaC2) in a strain of Pseudomonas fluorescens IBUN S1602 isolated from Colombian soil

La cepa Pseudomonas fluorescens IBUN S1602 conforma el grupo de aislamientos provenientes de suelos colombianos de caña de azúcar, que acumula polihidrioxialcanoato (PHA), fue seleccionada como promisoria para escalamiento comercial por tener afinidad por sustratos alternativos y económicos como el glicerol, aceites usados, suero de leche, entre otros. Dada la importancia de la enzima sintasa en la síntesis de los PHAs, en el presente trabajo se realizó el análisis molecular de los genes phaC1 y phaC2 que codifican las enzimas sintasas tipo II (PhaC1 y PhaC2). Para la obtención de los amplímeros requeridos en la secuenciación, se utilizó la técnica de PCR bajo condiciones estandarizadas para iniciadores diseñados reportados en las bases de datos. Se identificaron dos fragmentos de 1680 pb y 1683 pb correspondientes a phaC1 y phaC2. El análisis comparativo de las secuencias proteicas resultantes de estos genes demuestra que la sintasa IBUN S1602 contiene la región α/β hidrolasa y 8 residuos de aminoácidos conservados, que son características de las sintasas examinadas a nivel mundial. Se analizó la estructura enzimática a nivel primario y se predijo la secundaria. Se concluyó que las sintasas de la cepa Pseudomonas fluorescens IBUN S1602 presentan alta homología con las sintasas tipo II que se reportan para Pseudomonas. Los resultados obtenidos contribuyen al entendimiento básico de la biosíntesis de PHA, la cual permitirá, en un futuro, el aumento de la calidad de PHA debida a la modulación del nivel de sintasa que se exprese en un organismo recombinante, con el fin de variar el peso molecular del biopolímero, propiedad esencial en el estudio de aplicaciones industriales.

The strain Pseudomonas fluorescens IBUN S1602 forms the group of isolates from colombian sugarcane soil´s, which accumulates polyhydroxyalkanoate biopolymer (PHA) and was selected as promising for commercial scale by having affinity for economic and alternative substrates such as glycerol, oils, whey, among others. Given the importance of the synthase enzyme in the synthesis of PHAs, was realized the molecular analysis of genes phaC1 and phaC2 which encode type II synthases (PhaC1 y PhaC2). To obtain the amplimers required in the sequencing, was used the PCR technique under standardized conditions for primers designed based on the updated review in databases. Were identified two fragments of 1680 bp and 1683 bp for phaC1 and phaC2. Comparative analysis of the resulting protein sequences of these genes shows that the IBUN S1602 synthases containing the region α/β hydrolase and 8 conserved amino acid residues that are characteristic of synthases examined worldwide. Enzyme structure was analyzed at the primary level and was predicted the secondary. It is concluded that synthase strain Pseudomonas fluorescens IBUN S1602 has high homology with type II synthases that are reported for Pseudomonas. The results contribute to basic understanding of the biosynthesis of PHA, and will allow in the future, increasing the quality of PHA due to modulation of the level of synthase is expressed in a recombinant organism, in order to vary the weight molecular biopolymer, an essential property in the study of industrial applications.

Immune safety evaluation of polymerized porcine hemoglobin (pPolyHb): a potential red blood cell substitute.

Polymerized Porcine Hemoglobin (pPolyHb), a hemoglobin-based oxygen carrier (HBOC), was developed as a potential red blood substitute for clinical applications. Assessment of its effects on the immune system is an important component of the overall safety evaluation of HBOC. For this purpose, we assessed three inflammation indicators, including complement C3a, IL-6, and TNF-? in cultured cells and in a rat model when pPolyHb was incubated or administrated with the cells/animals. Our results suggested that the levels of these three indicators were not statistically changed upon pPolyHb stimulation, indicating that pPolyHb is not immunotoxic to cells and animals in this aspect.

Polymeric immunoglobulin receptor.

The surface of mucosal sites, such as the intestinal tract, are covered by epithelial cells. To protect the intestinal environment from invading pathogens and maintain homeostasis, the human body developed an exquisite acquired immune system, referred to as the mucosal immune system, in which epithelial cells and lymphocytes function cooperatively. The main player in this immune system is the polymeric immunoglobulins (pIgs), in particular dimeric IgA (dIgA). To exert its protective effect, dIgA produced in the lamina propria must be transported to the intestinal lumen across epithelial cells. This process is called transcytosis and is mediated by polymeric immunoglobulin receptor (pIgR), which is exclusively produced by intestinal epithelial cells (IECs). DIgA is captured by pIgR on the basolateral surface of IECs and transcytosed to the opposite side of IECs. The dIgA-pIgR complex is expressed on the apical surface of IECs and proteolytically cleaved to generate secretory IgA (SIgA). This review describes the current understanding and recent progress in this research field.

Immunomodulatory effects of Aureobasidium pullulans SM-2001 exopolymers on the cyclophosphamide-treated mice.

The immunomodulatory effects of exopolymers of Aureobasidium pullulans SM-2001 containing beta-1,3/1,6-glucan were evaluated on the cyclophosphamide (CPA)-treated mice. To induce immunosuppress, 150 and 110 mg/kg of CPA were intraperitoneally injected at 1 and 3 days before start of test material administrations, respectively. Exopolymers were subcutaneously or orally administered in a volume of 10 ml/kg, 4 times; 12-hr intervals from 24 hrs after second treatment of CPA. After treatment of exopolymers, the changes of thymus and spleen weights, splenic amounts of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-10, thymic and splenic CD3+, CD4+, CD8+ and TNF-alpha+ cells were monitored in CPA-treated mice. As results of CPA treatment, dramatical decreases of the CD3+, CD4+, CD8+ and TNF-alpha+ cells were detected in thymus and spleen with decreases of thymus and spleen weights. In addition, decreases of splenic TNF-alpha, IL-1beta and IL-10 contents were also detected at flow cytometrical observations. However, oral and subcutaneous treatment of exopolymers effectively reduced the immunosuppressive changes induced by CPA. Therefore, it is concluded that exopolymers of A. pullulans can be effectively prevent the immunosuppress mediated, at least partially, recruitment of T cells and TNF-alpha+ cells or enhancement of their activity, and can provide effective prevention or treat regimes for the immunosuppress and related diseases such as cancer, sepsis and high-dose chemotherapy or radiotherapy.

Histological study on side effects and tumor targeting of a block copolymer micelle on rats.

Histological examinations were performed with polymeric micelle-injected rats for evaluations of possible toxicities of polymeric micelle carriers. Weight of major organs as well as body weight of rats was measured after multiple intravenous injections of polymeric micelles forming from poly(ethylene glycol)-b-poly(aspartate) block copolymer. No pathological toxic side effects were observed at two different doses, followed only by activation of the mononuclear phagocyte system (MPS) in the spleen, liver, lung, bone marrow, and lymph node. This finding confirms the absence of--or the very low level of--in vivo toxicity of the polymeric micelle carriers that were reported in previous animal experiments and clinical results. Then, immunohistochemical analyses with a biotinylated polymeric micelle confirmed specific accumulation of the micelle in the MPS. The immunohistochemical analyses also revealed, first, very rapid and specific accumulation of the micelle in the vasculatures of tumor capsule of rat ascites hepatoma AH109A, and second, the micelle's scanty infiltration into tumor parenchyma. This finding suggests a unique tumor-accumulation mechanism that is very different from simple EPR effect-based tumor targeting.

An antigenic epitope of influenza virus nucleoprotein (NP) associated with polymeric forms of NP.

Intracellular influenza virus nucleoprotein (NP) is characterized by a high efficiency of homo-polymers formation, however their antigenic structure is still incompletely known. Herein, we report that RNase-resistant intracellular NP homo-polymers have a highly ordered conformational antigenic epitope, which depends on inter-subunit interactions of monomeric NPs. Our studies have shown that in radioimmunoprecipitation (RIPA) intracellular NP polymers bind mAb N5D3 and RNase does not prevent their mAb binding. In contrast to NP polymers, NP monomeric subunits, obtained by thermo-dissociation of NP polymers, fail to bind the mAb N5D3 in RIPA. At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope. The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation. Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration. The results obtained suggest that in vivo formation and in vitro renaturation of the N5D3 epitope depend on inter-subunit interactions of monomeric NPs and NP-NP interactions influence the antigenic structure of the influenza virus NP polymers.

Chemical characteristics and immuno-stimulating properties of biopolymers extracted from Acanthopanax sessiliflorus.

During our search for macrophage stimulating compounds from medicinal plants, we isolated biopolymers from Acanthopanax sessiliflorus. Isolated fraction AS-5 showed maximum potential, and stimulated lysosonal enzymatic activity by 230% at 300 microg/ml. The nitric oxide (NO) producing ability of AS-5 100 microg/ml was 58 microM when treated with interferon-gamma and lipopolysaccharide 20 micro/ml. The lymphocyte proliferating effects of isolated biopolymer fractions were also investigated. Highest lymphoproliferative activity (a 2.8-fold enhancement compared to salines treated group was exhibited by AS-3 at 200 micro/ml followed by AS-5 and AS-6. The AS-3 fraction stimulated only T-lymphocytes and had little or no effect on B-lymphocyte proliferation. Partially methylated alditol acetates were prepared to elucidate the glycosyl linkage-compositions of the AS-3 and AS-5 biopolymers, and were analyzed by GC-MS. The AS-3 and AS-5 biopolymer fractions were found to contain 2,3,4-tri-O-methyl-D-glucitol, 2,3,4-tri-O-methyl-D-galacitol 3,4,6-tri-O-methyl-galacitol, 2-O-methyl-arabinitol and 2,4,6-tri-O-methyl-D-glucitol, 2,3,6-tri-O-methyl-D-galacitol linkages, respectively.

A polymeric bacterial protein activates dendritic cells via TLR4.

The enzyme lumazine synthase from Brucella spp. (BLS) is a highly immunogenic protein that folds as a stable dimer of pentamers. It is possible to insert foreign peptides and proteins at the 10 N terminus of BLS without disrupting its general folding, and these chimeras are very efficient to elicit systemic and oral immunity without adjuvants. In this study, we show that BLS stimulates bone marrow dendritic cells from mice in vitro to up-regulate the levels of costimulatory molecules (CD40, CD80, and CD86) and major histocompatibility class II Ag. Furthermore, the mRNA levels of several chemokines are increased, and proinflammatory cytokine secretion is induced upon exposure to BLS. In vivo, BLS increases the number of dendritic cells and their expression of CD62L in the draining lymph node. All of the observed effects are dependent on TLR4, and clearly independent of LPS contamination. The described characteristics of BLS make this protein an excellent candidate for vaccine development.

A significant number of human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma interferon.

Despite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8(+) T lymphocytes are discussed.

Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens.

We have evaluated the ability of two carbohydrate biopolymers, chitosan and gellan, to enhance antibody responses to subunit influenza virus vaccines delivered to the respiratory tracts of mice. Groups of mice were vaccinated three times intranasally (i.n.) with 10 microg of purified influenza B/Panama virus surface antigens (PSAs), which consist of hemagglutinin (HA) and neuraminidase (NA), either alone or admixed with chitosan or gellan solutions. Separate groups were vaccinated subcutaneously (s.c.) with PSAs adsorbed to Alhydrogel or chitosan or gellan alone i.n. Serum antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) for influenza virus-specific immunoglobulin G (IgG) and by HA inhibition (HAI) and NA inhibition (NAI) assays. The local respiratory immune response was measured by assaying for influenza virus-specific IgA antibody in nasal secretions and by enumerating nasal and pulmonary lymphocytes secreting IgA, IgG, and IgM anti-influenza virus-specific antibodies by enzyme-linked immunospotting (ELISPOT). When administered alone i.n., B/Panama PSA was poorly immunogenic. Parenteral immunization with B/Panama PSA with Alhydrogel elicited high titers of anti-B/Panama antibodies in serum but a very poor respiratory anti-B/Panama IgA response. In contrast, i.n. immunization with PSA plus chitosan stimulated very strong local and systemic anti-B/Panama responses. Gellan also enhanced the local and serum antibody responses to i.n. PSA but not to the same extent as chitosan. The ability of chitosan to augment the immunogenicity of influenza vaccines given i.n. was confirmed using PSA prepared from an influenza A virus (A/Texas H1N1).

Purification and characterization of two mannan-binding lectins from mouse serum.

Mannan-binding lectin (MBL) is a serum protein that activates the complement system after binding to glycoconjugates found on the surface of microorganisms. By molecular cloning two forms of MBL have been identified in the mouse (mMBL-A and mMBL-C), but only mMBL-A has been purified and characterized at the protein level. MBL-C has been termed the liver form of MBL. The present report describes the purification and characterization of mMBL-A and mMBL-C from serum. The two forms of mMBL could be separated both by ion-exchange and carbohydrate-affinity chromatography. The initial identification by immunochemical technique was confirmed by N-terminal amino-acid sequencing. Both proteins give bands corresponding to polypeptide chains of 28 kDa on SDS-PAGE in the reduced state, but mMBL-A migrated more rapidly than mMBL-C in acid/urea-PAGE, in accordance with the calculated pIs. Both forms mediated activation of complement component C4 in mannan-coated microtiter wells. MBL-A showed a higher affinity for d -glucose and alpha-methyl-d -glucose then did MBL-C. Serum concentrations of mMBL-A in laboratory strains and wild mice were found to vary from 5 to 80 microg/ml, with wild mice tending to show higher levels than laboratory strains.

Rectal and vaginal immunization with a macromolecular multicomponent peptide vaccine candidate for HIV-1 infection induces HIV-specific protective immune responses.

An effective vaccine for human immunodeficiency virus (HIV) is needed to stimulate the immune response of the genital mucus to prevent mucosal transmission of the virus. We have developed a macromolecular multicomponent peptide vaccine candidate, VC1. Both rectal and vaginal immunization of VC1 mixed with cholera toxin (CT) induced HIV-1-specific IgA antibody in mouse fecal extract solution and vaginal wash. These antibody productions were enhanced by the combination with IL-4 or GM-CSF expressing plasmids. Either fecal extract or vaginal wash solution from immunized mice inhibited production of HIV-1IIIB p24 protein. The mononuclear cells from spleen, intestinal lymph nodes, or Peyer's patches from VC1- and CT-immunized mice released IFN-gamma or IL-4, when these cells were co-cultured with VC1 antigen. In addition, the regional lymphoid cells from rectal and vaginal region of mice immunized with VC1 and CT also elicited a substantial level of HIV-1-specific cytotoxic T cell (CTL) response. This CTL response was enhanced by the addition of IL-12 expressing plasmid. Our results clearly demonstrated that both rectal and vaginal immunization could induce systemic and mucosal immunities specific for HIV-1.

Extensive alanine substitutions increase binding affinity of an influenza nucleoprotein peptide to HLA-Aw68 and do not abrogate peptide-specific CTL recognition.

Class I MHC molecules bind peptides in the endoplasmic reticulum and present them at the cell surface to circulating CD8+ T cells for analysis. We have examined binding of peptides and stabilization of HLA-Aw68 class I molecules using synthetic peptide variants of an influenza virus nucleoprotein peptide, NP91-99 (KTGGPIYKR). We have demonstrated that insertion of increasing numbers of alanines in the center of the peptide (between P and I), to increase a natural bulging out of the peptide-binding cleft, results in a large decrease in thermal stability. Although there is a great decrease in the t 1/2 of the MHC/peptide complex for NP-1A compared with NP91-99, a T cell line, stimulated by NP91-99, recognizes NP-1A efficiently. Peptide variants with three or more alanines do not show saturable binding to HLA-Aw68 and also are not recognized by the T cell line. Thermal studies show that polyalanine peptides with minimal anchors and nearly all TCR contact residues exchanged stabilized HLA-Aw68 to high temperatures. Additionally, some of these polyalanine peptides are recognized by T cell lines generated against NP91-99. Analysis of the peptide sequences show that the stabilization effects are not due to the hydrophobicity of the peptide. These data suggest that the strength of binding of peptides to HLA-Aw68 is not only dictated by the presence of anchor residues but also by the lack of unfavorable contacts between the peptide ligand and class I MHC-binding cleft.

In vitro comparison of the biologic activities of monoclonal monomeric IgA, polymeric IgA, and secretory IgA.

Secretory IgA (S-IgA), a major humoral mediator of mucosal immunity, is a polymeric Ig containing an unusual extra polypeptide, secretory component (SC), added during transcytosis through epithelial cells. Polymeric S-IgA is more effective than monomeric IgA (mIgA) and IgG in neutralizing viruses. It is not known whether this increased efficacy is due solely to the polymeric structure of the molecule or whether SC itself makes S-IgA more efficient; a quantitative in vitro comparison of the biologic activities of S-IgA and pIgA has not been reported. We prepared purified pIgA and mIgA mAbs directed toward the H1 hemagglutinin of PR8 influenza virus and purified monoclonal S-IgA (made from monoclonal pIgA injected into a Lewis rat and collected as S-IgA from bile) and compared their abilities to carry out hemagglutination inhibition (HI) and neutralization of the infectivity of PR8 influenza virus in vitro. The polymeric Igs (pIgA and S-IgA) were 5 times more effective than mIgA in HI and 7 to 10 times more effective than mIgA in virus neutralization. Addition of SC to pIgA did not modify its ability to mediate HI and had only a minimal effect (S-IgA was 1.4 times more effective) on its ability to neutralize influenza virus in vitro. Trypsin preincubation partially abolished mIgA- or pIgA-mediated, but not S-IgA-mediated, viral neutralization. Thus, although S-IgA is more stable functionally than pIgA, the addition of SC does not influence, positively or negatively, the biologic activity associated with the Fab of S-IgA.

Analysis of the expression of peptide-major histocompatibility complexes using high affinity soluble divalent T cell receptors.

Understanding the regulation of cell surface expression of specific peptide-major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide-MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR-Ig) that have high affinity for their cognate peptide-MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR-Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus-1 presented by human histocompatibility leukocyte antigens-A2. Further, using 2C TCR- Ig, a more detailed analysis of the interaction with cognate peptide-MHC complexes revealed several interesting findings. Soluble divalent 2C TCR-Ig detected significant changes in the level of specific antigenic-peptide MHC cell surface expression in cells treated with gamma-interferon (gamma-IFN). Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of gamma-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide-MHC complexes. Analysis of the binding of 2C TCR-Ig for specific peptide-MHC ligands unexpectedly revealed that the affinity of the 2C TCR-Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8-H-2 Kbm3, is very low, weaker than 71 microM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca-H-2 Ld, is approximately 1000-fold higher. Thus, negatively selecting peptide-MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses.

Evidence that the two C1q binding membrane proteins, gC1q-R and cC1q-R, associate to form a complex.

Two types of widely coexpressed, highly acidic, cell membrane binding proteins that display preferential domain specificity for C1q have been described: a 60-kDa calreticulin homologue, designated cC1q-R, that binds to the collagen-like "stalk" and a 33-kDa glycoprotein with affinity for the globular "heads" (gC1q-R). Although the two molecules are known to be coexpressed on all cell types examined to date and often coelute during purification, there is no direct evidence showing that they associate with each other either on the membrane or when examined in a purified system. In this report we present the first evidence that 1) biotinylated cC1q-R binds to recombinant as well as native gC1q-R, as assessed by solid phase ELISA; 2) binding sites for cC1q-R are located within N-terminal residues 76 through 93 of the mature form of gC1q-R and within residues 204 through 218; 3) this interaction is inhibited by two mAbs, 60.11 and 46.23, that recognize primarily epitopes within the N terminus of gC1q-R corresponding to residues 74 through 96 and by mAb 74.5.2 that recognizes epitopes within residues 204 through 218; and 4) biotinylated cC1q-R binds to microtiter-fixed Raji and K562 cells, and this interaction is inhibited by mAb 60.11. Furthermore, coimmunoprecipitation analysis of Raji cell membranes with anti-gC1q-R mAbs showed the presence of cC1q-R in addition to gC1q-R. Taken together, the evidence suggests that cC1q-R is able to form a complex with gC1q-R and may associate with gC1q-R on the cell surface.