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At least 289 records · Page 16Linked to original sources
PubMed2004

Astroglial and microglial activation in the wistar rat ventral tegmental area after a single striatal injection of 6-hydroxydopamine.

Astroglial and microglial activation were analyzed in the ventral tegmental area (VTA) in adult male Wistar rats, after an unilateral striatal 6-hydroxydopamine (6-OHDA) injection. Different doses (8, 4, and 1 microg) of 6-OHDA were injected in the left side of the neostriatum; animals were sacrificed 22 days later. Control animals received an injection of the same volume of the solvent. The tyrosine hydroxylase (TH) positive dopamine cells, the glial fibrillary acidic protein (GFAP) immuno -labeled astrocytes, and the OX42 immunoreactive microglia were visualized by means of immunohistochemistry and quantified by stereologic methods employing the optical dissector and the point intercepts. The number and the density of TH immunoreactive cell bodies were decreased by 45% and 46%, respectively, in the sampled field of the ipsilateral VTA of 8 microg 6-OHDA injected rats. The GFAP immunohistochemistry revealed in the ipsilateral VTA increases the number and density of astroglial cells (154% and 166% of control, respectively) in the rats with a higher dose of the 6-OHDA, and also in the volume fraction of the astroglial processes after 8 microg (41% of control) and 4 microg (24% of control) of 6-OHDA. Increased number (76% of control) and density (77% of control) of OX42 microglial labeled profiles and microglial processes (51% of control) were found in the ipsilateral VTA of the 8 microg 6-OHDA injected animals. These results suggest that the retrograde degeneration of the mesostriatal dopamine pathways, induced by a striatal injection of 6-OHDA, leads to astroglial and microglial reactions in the VTA. The interaction between activated glial cells may be involved in the wounding and repair events in the partial lesioned system, and also in the trophic paracrine responses in the surviving VTA dopamine neurons.

Adrenergic Agents↗
PubMedDate not supplied

Increased extracellular matrix metalloproteinase inducer (EMMPRIN) expression in pulmonary fibrosis.

Extracellular matrix metalloproteinase inducer (EMMPRIN) was examined on bronchoalveolar lavage fluids (BALFs) and lung tissue from patients with fibrosis (usual interstitial pneumonia-idiopathic pulmonary fibrosis [UIP-IPF], n = 15; diffuse parenchymal lung diseases without IPF characteristics on computerized tomography scan, n = 8) and without fibrosis (n = 6). In UIP-IPF, EMMPRIN staining was increased in areas of fibrosis, mainly in macrophages and in epithelial cells. EMMPRIN was also found in the extracellular medium with significant levels in patients with lung fibrosis compared to subjects without fibrosis. Moreover, macrophages from patients with lung fibrosis spontaneously produce EMMPRIN. These findings show that EMMPRIN is increased in lung fibrosis.

Adolescent↗
PubMed2006

Immunolocalization of EMMPRIN (CD147) in the human eye and detection of soluble form of EMMPRIN in ocular fluids.

PURPOSE: To study the cellular distribution of extracellular matrix metalloproteinase inducer (EMMPRIN; CD147) in the human eye and the corneal and retinal pigment epithelium cell lines and its possible existence as a soluble protein in ocular fluids. METHODS: Immunohistochemistry was performed on human eyes and for cell cultures. Different EMMPRIN isoforms were analyzed by Western blotting in ocular fluids. RESULTS: EMMPRIN immunostaining could be detected in the corneal and conjunctival epithelium, the endothelium, and in the stromal keratocytes, the retinal pigment epithelium, several retinal layers and nerve fibers in the optic nerve head. Both cell lines deposit EMMPRIN on the cell membranes. Soluble EMMPRIN could also be detected in the tear fluid, aqueous humor, and vitreous samples in the form of multiple immunoreactive species. CONCLUSIONS: EMMPRIN is specifically expressed in the human eye only by certain tissue structures, thus suggesting specialized functions. The protein also exists naturally in soluble forms in ocular fluids representing presumably monomeric and multimeric forms, a notion that confirm and further extends its previously known role mainly as a transmembrane protein. These findings suggest that EMMPRIN can regulate not only cell surface functions in the human eye but also certain peri- and extracellular functions.

Adult↗
PubMed2006

Deglycosylation of CD147 down-regulates Matrix Metalloproteinase-11 expression and the adhesive capability of murine hepatocarcinoma cell HcaF in vitro.

CD147 is a plasma membrane glycoprotein, enriched on the surface of many malignant tumor cells. As a result of heterogeneous N-glycosylation, CD147 exists in both a highly glycosylated form, HG-CD147 ( approximately 40-60 kDa) and lowly glycosylated form, LG-CD147 ( approximately 32 kDa). This experiment investigated the possible role of CD147 glycosylation in the HcaF, HcaP and Hepa1-6 mouse hepatocarcinoma cell lines, which have high, low and no metastatic potential in the lymph nodes. Western blot analysis showed that the ratio of HG-CD147/LG-CD147 protein expression on HcaF and HcaP were much higher than that on Hepa1-6 cells. By treatment with tunicamycin (TM), an inhibitor of N-glycosylation, the expression level of HG-CD147 decreased and the LG-CD147 disappeared completely in HcaF cells. Meanwhile, Matrixmetallproteinase-11 (MMP-11) protein expression was down-regulated, and the adhesive capability of HcaF cells to endothelial cells in cryosection of mouse lymph nodes decreased. These results indicated that the glycosylation of CD147 plays a crucial role. It is HG-CD147 that may contribute more to tumor progress, invasion and metastasis into lymph node rather than LG-CD147. The results of this study are of biological and clinical importance.

Animals↗
PubMed1993

Breaching the diffusion barrier that compartmentalizes the transmembrane glycoprotein CE9 to the posterior-tail plasma membrane domain of the rat spermatozoon.

CE9 is a posterior-tail domain-specific integral plasma membrane glycoprotein of the rat testicular spermatozoon. During epididymal maturation, CE9 undergoes endoproteolytic processing and then redistributes into the anterior-tail plasma membrane domain of the spermatozoon (Petruszak, J. A. M., C. L. Nehme, and J. R. Bartles. 1991. J. Cell. Biol. 114:917-927). We have determined the sequence of CE9 and found it to be a Type Ia transmembrane protein identical to the MRC OX-47 T-cell activation antigen, a member of the immunoglobulin superfamily predicted to have two immunoglobulin-related loops and three asparagine-linked glycans disposed extracellularly. Although encoded by a single gene and mRNA in the rat, the majority of spermatozoal CE9 is of smaller apparent molecular mass than its hepatocytic counterpart due to the under-utilization of sites for asparagine-linked glycosylation. By fluorescence recovery after photobleaching, CE9 was determined to be mobile within the posterior-tail plasma membrane domain of the living rat testicular spermatozoon, thus implying the existence of a regional barrier to lateral diffusion that is presumed to operate at the level of the annulus. Through the development of an in vitro system, the modification of this diffusion barrier to allow for the subsequent redistribution of CE9 into the anterior-tail domain was found to be a time-, temperature-, and energy-dependent process.

Amino Acid Sequence↗
PubMed1998

Apical polarity of N-CAM and EMMPRIN in retinal pigment epithelium resulting from suppression of basolateral signal recognition.

Retinal pigment epithelial (RPE) cells apically polarize proteins that are basolateral in other epithelia. This reversal may be generated by the association of RPE with photoreceptors and the interphotoreceptor matrix, postnatal expansion of the RPE apical surface, and/or changes in RPE sorting machinery. We compared two proteins exhibiting reversed, apical polarities in RPE cells, neural cell adhesion molecule (N-CAM; 140-kD isoform) and extracellular matrix metalloproteinase inducer (EMMPRIN), with the cognate apical marker, p75-neurotrophin receptor (p75-NTR). N-CAM and p75-NTR were apically localized from birth to adulthood, contrasting with a basolateral to apical switch of EMMPRIN in developing postnatal rat RPE. Morphometric analysis demonstrated that this switch cannot be attributed to expansion of the apical surface of maturing RPE because the basolateral membrane expanded proportionally, maintaining a 3:1 apical/basolateral ratio. Kinetic analysis of polarized surface delivery in MDCK and RPE-J cells showed that EMMPRIN has a basolateral signal in its cytoplasmic tail recognized by both cell lines. In contrast, the basolateral signal of N-CAM is recognized by MDCK cells but not RPE-J cells. Deletion of N-CAM's basolateral signal did not prevent its apical localization in vivo. The data demonstrate that the apical polarity of EMMPRIN and N-CAM in mature RPE results from suppressed decoding of specific basolateral signals resulting in randomized delivery to the cell surface.

Animals↗
PubMed2005

Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus.

To identify the function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome (SARS) coronavirus (CoV), we analyzed the protein-protein interaction among HAb18G/CD147, cyclophilin A (CyPA), and SARS-CoV structural proteins by coimmunoprecipitation and surface plasmon resonance analysis. Although none of the SARS-CoV proteins was found to be directly bound to HAb18G/CD147, the nucleocapsid (N) protein of SARS-CoV was bound to CyPA, which interacted with HAb18G/CD147. Further research showed that HAb18G/CD147, a transmembrane molecule, was highly expressed on 293 cells and that CyPA was integrated with SARS-CoV. HAb18G/CD147-antagonistic peptide (AP)-9, an AP of HAb18G/CD147, had a high rate of binding to 293 cells and an inhibitory effect on SARS-CoV. These results show that HAb18G/CD147, mediated by CyPA bound to SARS-CoV N protein, plays a functional role in facilitating invasion of host cells by SARS-CoV. Our findings provide some evidence for the cytologic mechanism of invasion by SARS-CoV and provide a molecular basis for screening anti-SARS drugs.

Antigens, CD↗
PubMed2001

Cellular inflammatory response associated with breakdown of the blood-brain barrier after closed head injury in rats.

This study reports a widespread microglial response characterized by an upregulation of surface antigens, such as complement type 3 receptors (CR3) and major histocompatibility complex (MHC) class II antigens on these cells following closed head injury. Increased expression of CR3 (OX-42) and MHC class II antigens (OX-6) was observed in rats killed at 1, 3, and 5 days after injury. Intense OX-42 immunoreactivity was observed in microglial cells throughout the brain with a smaller number of them being OX-6 positive. In addition to microglial reaction, astrocytic activation reflected in cellular hypertrophy and increased immunoreactivity for glial fibrillary acidic protein (GFAP) was observed at 5 days after head injury. Together with the above, a diffuse perivascular and intraneuronal immunostaining for immunoglobulin G (IgG) was observed primarily in the cerebral cortex. This was accompanied by an enhanced expression of both endothelial nitric oxide synthase (eNOS) in blood vessels and inducible nitric oxide synthase (iNOS) in brain macrophages. In rats subjected to closed head injury followed by a single intraperitoneal (i.p.) injection of rhodamine isothiocyanate (RhIc), seepage of the fluorescent dye into the neuropil was observed. This had resulted in the labelling of the cortical neurons clearly demonstrating a breakdown of the blood-brain barrier (BBB). In the latter, it is conceivable that the ensuing leakage of plasma immunoglobulins and other serum-derived materials could induce the expression of MHC class II antigens on microglia. The mechanism causing the BBB dysfunction is not clear, although present results suggest that excessive release of nitric oxide (NO) may be a contributory factor. The widespread activation of microglia in rats after head injury suggests their involvement in increased endocytosis and immunological responses.

Animals↗
PubMed2002

Production of antibodies by single DNA immunization: comparison of various immunization routes.

DNA immunization is a recent vaccination method that induces humoral and cellular immune responses in a range of hosts. Different immunization routes induce a different degree of the immune response. In the present report, we demonstrate that multiple intramuscular immunizations of plasmid DNA encoding various leukocyte surface molecules induced a specific antibody response. In contrast, a single intramuscular immunization could not induce antibody production. To study the induction of antibody response after a single immunization of plasmid DNA, mice were single-dose intramuscularly, intraperitoneally, intravenously and intrasplenically immunized, simultaneously, with the same preparation of plasmid DNA encoding CD147 membrane protein. We observed that only the intrasplenic route induced specific antibody production. The induction of antibody by intrasplenic immunization was confirmed by using plasmid DNA encoding CD54 molecule. By this single-dose DNA intrasplenic immunization, the generated antibodies could be detected in mice up to 6 months. These results suggest that the injected DNA is expressing the relevant protein antigen in the spleen for several months after injection. Our results demonstrate that direct immunization of antigen-encoding DNA into the spleen is a more effective method for induction of antibody production. This finding may support future investigations of DNA vaccination strategies that specifically promote the uptake of plasmid by splenocytes. Intrasplenic immunization may also be helpful in the understanding of the early events of the immune response to DNA vaccine and be useful as an effective route for the induction of immune responses.

Animals↗
PubMed2006

Regulation of matrix metalloproteinase production and tumor cell invasion by four monoclonal antibodies against different epitopes of HAb18G/CD147 extracellular domain.

HAb18G/CD147, a membrane spanning molecule and highly expressed in hepatocellular carcinoma (HCC) cells, was shown to stimulate the production of matrix metalloproteinases (MMPs) in the interaction of tumor cells and fibroblasts. Studies on the EMMPRIN/CD147 showed that CD147 extracellular domain is involved in the induction of MMPs. To study the biological molecular function of HAb18G/CD147 extracellular domain (HAb18G/CD147-ED) on production of MMPs following mediated tumor cell invasion, we isolated four novel monoclonal anibodies (MAbs)-1B3, 3B3, HAb18Gedomab1, and HAb18Gedomab2-against HAb18G/CD147-ED by immunization of BALB/c mice with purified HAb18G/CD147-ED fragments, which were efficiently expressed in Escherichia coli. Gelatin zymography and Boyden chamber assays were used to identify the production of MMPs in the co-cultured human fibroblast and HCC cells, and to quantify the migrated cells in the presence of the generated MAbs. The results showed that two MAbs (1B3 and 3B3) inhibited [corrected] the secretion of MMP-2 and [corrected] the HCC cell invasion, whereas the other two MAbs (HAb18Gedomab1 and HAb18Gedomab2) had reverse function [corrected] FCM additive assay showed that four MAbs recognized different epitopes of HAb18G/CD147-ED. Taken together, the results suggest that various regions of HAb18G/CD147-ED participated in the regulation of MMP secretion.

Animals↗
PubMed1999

P-selectin blockade following fluid-percussion injury: behavioral and immunochemical sequelae.

Traumatic brain injury (TBI) can cause polymorphonuclear leukocyte (PMN) migration into brain parenchyma, mediating various cytodestructive mechanisms. We examined the effect of blocking leukocyte/endothelial cell adhesion molecules (CAMs) on the anatomic and behavioral sequelae in lateral fluid-percussion injury in rats. Monoclonal antibodies (MAb) directed against a functional (PB1.3) or nonfunctional (PNB1.6) epitope on endothelial P-selectin were used as treatments. Subjects were tested in the Morris water maze (MWM) at 7 and 14 days postinjury then immunohistochemistry was performed using antibodies that recognize ChAT, GFAP and OX-42. A second set of animals underwent myeloperoxidase (MPO) assay in the brain parenchyma and a third set was used to examine neutrophil migration using the MAb RP-3. Time in quadrant, but not escape latency or proximity improved with PB1.3 (p < 0.05). Similarly, PB1.3 reduced MPO levels after injury (p < 0.05), in the ipsilateral cortex. No significant difference occurred in neutrophil counts in cortex, corpus callosum, hippocampus, and thalamus between injured only rats and injured rats treated with PB1.3. Quantitative analysis of cholinergic cells in the medial septum showed a protective effect by PB1.3. Densitometry readings of GFAP and OX-42 immunolabeling revealed no discernible differences between the treated and untreated injured rats. Qualitatively, there was no difference in microglia or astrocyte response to treatment. Treatment with P-selectin blockade in brain-injured rats may reduce PMN migration into brain, help preserve cholinergic immunolabeling of medial septal nucleus neurons, and may alleviate mnemonic deficits.

Animals↗
PubMed2000

Rapid and widespread microglial activation induced by traumatic brain injury in rat brain slices.

In order to assess the role of circulating blood in early microglial activation after traumatic brain injury (TBI), controlled cortical impact injury was applied to adult rat brain slices (400 microm in thickness) and the microglial response was examined. The complement receptor (CR3) expression and morphological transformation of the microglia were evaluated by OX42 immunohistochemistry. At 5 min following injury, activated microglia with intense CR3 expression appeared throughout the hemisphere on the injured side. In contrast, the morphology and CR3 expression of the microglia on the contralateral side were indistinguishable from those of the resident ramified microglia seen in normal brains. At 30 min following injury, microglial activation was more pronounced on the injured side, while the microglia on the contralateral side still retained a ramified morphology. These results are consistent with our previous observations made in in vivo experiments, which indicate that, as the brain slice paradigm excludes variables arising from the circulating blood, the rapid and widespread microglial activation observed following TBI can not be attributed exclusively to the infiltration of blood-borne macrophages or molecules. Rather this activation is most likely caused by intrinsic mechanisms within the brain tissue, such as traumatic depolarization.

Animals↗
PubMed2006

Transplantation of fibroblasts that overexpress matrix metalloproteinase-3 into the site of spinal cord injury in rats.

Growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) are upregulated during glial scar formation at the site of spinal cord injury (SCI) in adult mammals. This CSPG-containing glial scar inhibits axonal regeneration. Matrix metalloproteinases (MMPs) can degrade CSPGs and other inhibitory proteins to promote neurite outgrowth. Increased MMP synthesis and secretion are observed in fibroblasts adjacent to tumor cells that express the protein EMMPRIN (Extracellular Matrix MetalloPRoteinase INducer). EMMPRIN transduction of cells provides an avenue to deliver increased levels of MMPs to the site of SCI in a sustained, localized, and moderate fashion. We explored the use of EMMPRIN-transduced cells as a mechanism to degrade CSPGs, facilitate axonal growth and improve recovery after SCI. Human dermal fibroblasts infected with a recombinant EMMPRIN adenovirus significantly increased secretion of MMP-3 compared to fibroblasts infected with a control adenovirus. Decreased CSPG immunoreactivity was observed in injured spinal cord sections when they were incubated with media from EMMPRIN-transduced fibroblasts. Conditioned media from EMMPRIN-transduced fibroblasts increased the length of neurites that were grown on a CSPG substrate. Rats that received contusive SCI and EMMPRIN-transduced fibroblast transplants demonstrated improved locomotor recovery compared to rats that received control fibroblasts, but not compared to other control groups. EMMPRIN-transplanted rats showed a significant increase in the number of retrogradely labeled cell bodies within brainstem nuclei and an increase in serotonergic fibers distal to the site of injury. EMMPRIN, and consequently MMP, delivery to the injured spinal cord may prove to be beneficial in reducing some of the physical barriers to axonal growth after SCI.

Adenoviridae↗
PubMed2004

The basolateral targeting signal of CD147 (EMMPRIN) consists of a single leucine and is not recognized by retinal pigment epithelium.

CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This elicited our interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, we carried out systematic site-directed mutagenesis to delineate basolateral targeting information in CD147. Our detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. We ruled out the involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, our study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells.

Adaptor Protein Complex 1↗
PubMed2004

Links between CD147 function, glycosylation, and caveolin-1.

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-catalyzed, beta1,6-branched, polylactosamine-type sugars, which account for its excess size. Therefore, CD147, which is itself elevated on invasive tumor cells, may make a major contribution to the abundance of beta1,6-branched polylactosamine sugars that appear on invasive tumor cells. It was shown previously that caveolin-1 associates with CD147, thus inhibiting CD147 self-aggregation and MMP induction; now we show that caveolin-1 associates with LG-CD147 and restricts the biosynthetic conversion of LG-CD147 to HG-CD147. In addition, HG-CD147 (but not LG-CD147) was preferentially captured as a multimer after treatment of cells with a homobifunctional cross-linking agent and was exclusively recognized by monoclonal antibody AAA6, a reagent that selectively recognizes self-associated CD147 and inhibits CD147-mediated MMP induction. In conclusion, we have 1) determined the biochemical basis for the unusual size variation in CD147, 2) established that CD147 is a major carrier of beta1,6-branched polylactosamine sugars on tumor cells, and 3) determined that caveolin-1 can inhibit the conversion of LG-CD147 to HG-CD147. Because it is HG-CD147 that self-aggregates and stimulates MMP induction, we now have a mechanism to explain how caveolin-1 inhibits these processes. These results help explain the previously established tumor suppressor functions of caveolin-1.

Amino Sugars↗
PubMed2000

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression.

CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin-like domains and a single charge-containing transmembrane domain. Here we use co-immunoprecipitation and chemical cross-linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton-linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2-CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co-localize, concentrating at the t-tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross-linking with anti-CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into 'caps'. In MCT-transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co-transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.

Animals↗
PubMed1999

T cell activation-associated epitopes of CD147 in regulation of the T cell response, and their definition by antibody affinity and antigen density.

CD147 is a broadly expressed cell surface glycoprotein of the Ig superfamily whose expression is up-regulated upon T cell activation. In order to elucidate a possible role of CD147 in T cell biology, we established 15 specific mAb. Seven distinct epitopes were defined by the mAb panel. Most of the mAb bound only to phytohemagglutinin (PHA)-activated but not resting T cells. We demonstrate that this was not because of true expression of activation-dependent neoepitopes but rather due to bivalent binding of the relatively low-affinity mAb (affinity constant KA values between 2.25 x 10(8) and 7 x 10(9) M-1) to the more densely expressed and/or more clustered CD147 molecules on the activated T cells. In contrast, the mAb with higher affinity (KA > 7 x 10(9) M-1) could stably bind in a monovalent fashion even to the relatively low dense CD147 molecules on resting T cells. This model might more generally explain the nature of 'activation epitopes' described previously in other leukocyte surface molecules. Finally, we provide evidence that induction of ordered dimerization of CD147 by a mAb directed to a unique epitope results in strong inhibition of CD3-mediated T cell activation.

Animals↗
PubMed1992

The hinge region of the CD8 alpha chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains.

The lymphocyte surface CD8 antigen is a heterodimer with each chain containing a single Ig-related domain, a hinge-like sequence, a transmembrane segment, and a short cytoplasmic sequence. A soluble form of the rat CD8 alpha chain was produced by introducing a stop codon into the cDNA at the end of the region encoding the extracellular sequence and expressed in Chinese hamster ovary cells. sCD8 alpha was produced at 20 mg/l, and consisted of monomers, dimers, and higher aggregates. The latter could be minimized, but not eliminated, by removal of one of the two cysteine residues in the hinge region by mutation and by growth in serum-free medium. The positions of the N- and O-linked glycosylation sites and the disulphide bond in the Ig-like domain were determined. The MRC OX-8 antibody was shown to react with a region from the CD8 alpha hinge containing 24 amino acids and the antigenic determinant was sensitive to neuraminidase digestion. A construct encoding the Ig-like domain of rat CD8 alpha without the hinge was not expressed in CHO cells, indicating the importance of the hinge region for expression. It seemed possible that the CD8 alpha hinge might facilitate expression of other Ig-related domains and such expression could be detected using the MRC OX-8 antibody. To test the system cDNA constructs were made with the rat CD8 alpha hinge spliced to the V-like domain of mouse CD8 alpha, to the V alpha and V beta domains of a T lymphocyte antigen receptor, and to one or both of the Ig-like domains of the MRC OX-47 membrane antigen. All these forms were expressed as soluble proteins that were detected with the MRC OX-8 antibody. This method may prove useful for the expression of Ig superfamily domains for raising antibodies and other studies.

Aged↗