Role of class B scavenger receptor type I in phagocytosis of apoptotic rat spermatogenic cells by Sertoli cells

Rat Sertoli cells phagocytose apoptotic spermatogenic cells, which consist mostly of spermatocytes, in primary culture by recognizing phosphatidylserine (PS) exposed on the surface of degenerating spermatogenic cells. We compared the mode of phagocytosis using spermatogenic cells at different stages of spermatogenesis. Spermatogenic cells were separated into several groups based on their ploidy, with purities of 60-90%. When the fractionated spermatogenic cell populations were subjected to a phagocytosis assay, cells with ploidies of 1n, 2n, and 4n were almost equally phagocytosed by Sertoli cells. All the cell populations exposed PS on the cell surface, and phagocytosis of all cell populations was similarly inhibited by the addition of PS-containing liposomes. Class B scavenger receptor type I (SR-BI), a candidate for the PS receptor, was detected in Sertoli cells. Overexpression of the rat SR-BI cDNA increased the PS-mediated phagocytic activity of Sertoli cell-derived cell lines. Moreover, phagocytosis of spermatogenic cells by Sertoli cells was inhibited in the presence of an anti-SR-BI antibody. Finally, the addition of high density lipoprotein, a ligand specific for SR-BI, decreased both phagocytosis of spermatogenic cells and incorporation of PS-containing liposomes by Sertoli cells. In conclusion, SR-BI functions at least partly as a PS receptor, enabling Sertoli cells to recognize and phagocytose apoptotic spermatogenic cells at all stages of differentiation.     

Involvement of mitogen-activated protein kinases in class B scavenger receptor type I-induced phagocytosis of apoptotic cells

Class B scavenger receptor type I (SR-BI) is a multiligand membrane protein expressed in a variety of cell types. This receptor is responsible for the incorporation of lipids from high density lipoprotein (HDL) by steroidogenic cells, as well as for the phosphatidylserine (PS)-mediated phagocytosis of apoptotic cells by some phagocytic cell types, such as testicular Sertoli cells. Although SR-BI directly binds to PS present on the surface of apoptotic cells, as to whether SR-BI transmits signals to induce engulfment has not been clear. In the present study, we examined this issue using a monoclonal antibody that neutralizes SR-BI activity and a chemical known to be an inhibitor of the SR-BI-mediated incorporation of HDL lipids. The chemical compound inhibited the incorporation of HDL lipids and PS-containing liposomes by an SR-BI-expressing culture cell line, with no effect on the binding of these targets. Similarly, the phagocytosis of PS-exposing apoptotic cells by primary cultured rat Sertoli cells was inhibited in the presence of either reagent, not at the recognition but at the engulfment step. The addition of apoptotic cells or PS-containing liposomes caused a temporal increment of the phosphorylation of all three mitogen-activated protein kinases, p38, extracellular-signal-regulated kinase (ERK) and c-Jun amino-terminal kinase (JNK), in Sertoli cells. The increase of phosphorylated p38 and ERK, but not of phosphorylated JNK, was cancelled in the presence of the monoclonal antibody. Furthermore, the level of Sertoli cell phagocytosis of PS-exposing apoptotic cells, as well as that of PS-containing liposomes, was reduced only when the actions of p38 and ERK were simultaneously repressed. In conclusion, these results indicate that SR-BI, when it binds to PS, transmits signals to activate the mitogen-activated protein kinase pathway, which leads to the induction of the engulfment of PS-exposing apoptotic cells by phagocytic cells.     

Expression and function of class B scavenger receptor type I on both apical and basolateral sides of the plasma membrane of polarized testicular Sertoli cells of the rat

Class B scavenger receptor type I (SR-BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR-BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR-BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle-stimulating hormone (FSH). When the expression of SR-BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first-round spermatogenesis and continued to be detectable thereafter. The level of SR-BI mRNA expression in Sertoli cells was lower at spermatogenic stages I-VI than at other stages. SR-BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR-BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR-BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR-BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.     

The predominance of one of the SR-BI isoforms is associated with increased esterified cholesterol levels not apoptosis in mink testis

Scavenger receptor class B type I (SR-BI) contributes to HDL-mediated cellular cholesterol efflux and is a phagocytosis-inducing phospholipid phosphatidylserine receptor in rat Sertoli cells, whereas the spliced variant of the SR-B gene, SR-BII, is implicated in the efflux of free cholesterol in macrophages. This study aimed to assess whether spontaneous autoimmune orchitis (AIO), which causes impaired clearance of apoptotic germ cells and spermatogenic arrest, involves SR-BI, SR-BII, and/or cholesterol. The levels measured during development and the annual reproductive cycle in normal mink were compared with those in mink with spontaneous AIO. Time periods with lowest tubular esterified cholesterol (EC) levels showed maximal SR-BI and SR-BII levels, and the periods when one or the other SR-BI isoform predominated showed increased EC levels and spermatogenic arrest in normal mink seminiferous tubules. In tubules with AIO, the predominance of only one or the other SR-BI isoform was the reverse of that measured in normal tubules, and it was associated with an increase in EC levels but not with apoptosis levels. SR-BI and SR-BII levels were not correlated with serum testosterone levels. SR-BI mainly localized to the Leydig cell, germ cell, and Sertoli cell surface, where its distribution was stage-specific. SR-BII was principally intracellular. Tubules from testes with AIO showed a deregulation of cholesterol homeostasis and SR-BI expression but relatively unchanged apoptosis levels. These results suggest that the expression of both SR-BI isoforms is required for the maintenance of low EC levels and that the predominance of only one isoform is associated with the accumulation of EC but not with apoptosis in the tubules.     

In vivo analysis of phagocytosis of apoptotic cells by testicular Sertoli cells

Sertoli cells, a somatic cell type present within the seminiferous tubules of testes, are responsible for the phagocytic elimination of apoptotic spermatogenic cells. We here established an in vivo assay system that enables us to quantitatively analyze Sertoli cell phagocytosis of apoptotic cells in testes of live mice. Apoptotic cells were injected into the seminiferous tubules of spermatogenic cell-depleted mice, and the occurrence of phagocytosis by Sertoli cells was examined by histochemically analyzing testis sections or dispersed testicular cells. We reproducibly observed similar levels of phagocytosis in either examination, and the ratio of Sertoli cells that engulfed injected apoptotic cells was almost the same between the two examinations. These results indicated that a quantitative in vivo assay system was established using the seminiferous tubules of live mice as 'test tubes.' We then determined the requirements for Sertoli cell phagocytosis of apoptotic cells using this assay. For this purpose, apoptotic cells were injected together with various phagocytosis inhibitors, and the extent of phagocytosis by Sertoli cells was determined. The results revealed that Sertoli cells phagocytose apoptotic cells in a manner dependent on class B scavenger receptor type I (SR-BI) of Sertoli cells and phosphatidylserine exposed at the surface of target cells, as previously observed in vitro using primary cultures of dispersed rat testicular cells. Furthermore, the amount of SR-BI in Sertoli cells increased after injection of apoptotic cells into the seminiferous tubules, suggesting a positive feedback regulation of the expression of this phagocytosis receptor.     

Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility

Many differentiating spermatogenic cells die by apoptosis during the process of mammalian spermatogenesis. However, very few apoptotic spermatogenic cells are detected by histological examination of the testis, probably due to the rapid elimination of dying cells by phagocytosis. Previous in vitro studies showed that Sertoli cells selectively phagocytose dying spermatogenic cells by recognizing the membrane phospholipid phosphatidylserine (PS), which is exposed to the surface of spermatogenic cells during apoptosis. We examined here whether PS-mediated phagocytosis of apoptotic spermatogenic cells occurs in vivo. For this purpose, the PS-binding protein annexin V was microinjected into the seminiferous tubules of normal live mice, and their testes were examined. The injection of annexin V caused no histological changes in the testis, but significantly increased the number of apoptotic spermatogenic cells as assessed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. The number of Sertoli cells did not change in the annexin V-injected testes, and annexin V itself did not induce apoptosis in primary cultured spermatogenic cells. These results indicate that annexin V inhibited the phagocytic clearance of apoptotic spermatogenic cells and suggest that PS-mediated phagocytosis of those cells occurs in vivo. Furthermore, the injection of annexin V into the seminiferous tubules brought about a significant reduction in the number of spermatogenic cells and epididymal sperm in anticancer drug-treated mice. This suggests that the elimination of apoptotic spermatogenic cells is required for the production of sperm.     

Binding of recombinant feline immunodeficiency virus surface glycoprotein to feline cells: role of CXCR4, cell-surface heparans, and an unidentified non-CXCR4 receptor

To address the role of CXCR4 in the cell-surface attachment of the feline immunodeficency virus (FIV), a soluble fusion protein, gp95-Fc, consisting of the surface glycoprotein (SU, gp95) of either a primary (PPR) or cell line-adapted (34TF10) FIV strain was fused in frame with the Fc domain of human immunoglobulin G1. The recombinant SU-immunoadhesins were used as probes to investigate the cellular binding of FIV SU. In agreement with the host cell range properties of both viruses, binding of 34TF10 gp95-Fc was observed for all cell lines tested, whereas PPR gp95-Fc bound only to primary feline T cells. 34TF10 gp95-Fc also bound to Jurkat and HeLa cells, consistent with the ability of FIV to use human CXCR4 as a fusion receptor. As expected, 34TF10 gp95-Fc binding to Jurkat cells was blocked by addition of stromal cell-derived factor 1alpha (SDF-1alpha), as was binding to the 3201 feline lymphoma cell line. However, SDF-1alpha, RANTES, macrophage inflammatory protein 1beta, and heparin all failed to inhibit the binding of either gp95-Fc to primary T cells, suggesting that a non-CXCR4 receptor is involved in the binding of FIV SU. In this regard, an unidentified 40-kDa protein species from the surface of primary T cells but not Jurkat and 3201 cells specifically coprecipitated with both gp95-Fc. Yet another type of binding of 34TF10 gp95-Fc to adherent kidney cells was noted. SDF-1alpha failed to block the binding of 34TF10 gp95-Fc to either HeLa, Crandel feline leukemia, or G355-5 cells. However, binding was severely impaired in the presence of soluble heparin, as well as after enzymatic removal of surface heparans or on cells deficient in heparan expression. These overall findings suggest that in addition to CXCR4, a non-CXCR4 receptor and cell-surface heparans also play an important role in FIV gp95 cell surface interactions on specific target cells.     

Stimulatory effects of B7-related protein-1 on cellular and humoral immune responses in mice

Inducible costimulator (ICOS) and B7-related protein-1 (B7RP-1) constitute a receptor-ligand pair involved in T cell costimulation. In this study, the stimulatory effects of B7RP-1 on cellular and humoral immune responses were investigated giving mice a construct with the extracellular domain of murine B7RP-1 fused with human IgG1 Fc (B7RP-1-Fc). B7RP-1-Fc stimulated contact hypersensitivity (CH) given near either the time of sensitization or challenge with oxazolone. When given near challenge time, B7RP-1-Fc stimulated CH more than a construct containing the extracellular domain of murine B7.2 and Fc (B7.2-Fc). B7RP-1-Fc increased the number of cells in lymph nodes draining the skin sensitized with oxazolone, especially activated T cells. B7RP-1-Fc also increased the ability of the cells in these lymph nodes to induce CH when transfused into naive mice. B7RP-1-Fc stimulated the production of anti-keyhole limpet hemocyanin (KLH) Ab, increasing anti-KLH IgG, IgG2a, and IgE, whereas B7.2-Fc did not affect this production. B7RP-1-Fc also increased the number of cells in lymph nodes draining the skin immunized with KLH and their production of IFN-gamma, IL-4, and IL-10 in response to KLH. Finally, B7RP-1-Fc increased the presence of eosinophils in the bronchoalveolar lavage and lungs of mice sensitized and challenged with OVA so to mount an asthmatic reaction. B7RP-1-Fc stimulates both cellular and humoral immune responses in vivo by increasing number and function of T and B cells reacting to Ag exposure.     

Alteration of hepatitis A virus (HAV) particles by a soluble form of HAV cellular receptor 1 containing the immunoglobin-and mucin-like regions

Hepatitis A virus (HAV) infects African green monkey kidney cells via HAV cellular receptor 1 (havcr-1). The ectodomain of havcr-1 contains an N-terminal cysteine-rich immunoglobin-like region (D1), followed by a mucin-like region that extends D1 well above the cell surface. D1 is required for binding of HAV, and a soluble construct containing D1 fused to the hinge and Fc portions of human immunoglobin G1 (IgG1), D1-Fc, bound and neutralized HAV inefficiently. However, D1-Fc did not alter the virions. To determine whether additional regions of havcr-1 are required to trigger uncoating of HAV, we constructed D1muc-Fc containing D1 and two-thirds of the mucin-like region fused to the Fc and hinge portions of human IgG1. D1muc-Fc neutralized 10 times more HAV than did D1-Fc. Sedimentation analysis in sucrose gradients showed that treatment of HAV with 20 to 200 nM D1muc-Fc disrupted the majority of the virions, whereas treatment with 2 nM D1muc-Fc had no effect on the sedimentation of the particles. Treatment of HAV with 100 nM D1muc-Fc resulted in low-level accumulation of 100- to 125S particles. Negative-stain electron microscopy analysis revealed that the 100- to 125S particles had the characteristics of disrupted virions, such as internal staining and diffuse edges. Quantitative PCR analysis showed that the 100- to 125S particles contained viral RNA. These results indicate that D1 and the mucin-like region of havcr-1 are required to induce conformational changes leading to HAV uncoating.     

Scavenger receptor class B type I as a receptor for oxidized low density lipoprotein

Scavenger receptor class B type I (SR-BI) has been established as the primary mediator of the selective transfer of lipids from HDL to mammalian cells. In addition to its role in cholesterol metabolism, SR-BI has been shown to bind apoptotic cells and thus could in theory also function as a scavenger receptor. We now show that SR-BI binds oxidized LDL (OxLDL) with high affinity (K(d) of 4.0 +/- 0.5 microg/ml) and mediates internalization and degradation to an extent comparable to that of other scavenger receptors, when normalized to binding activity. The best competitors for OxLDL binding to SR-BI were oxidized lipoproteins, whereas native or acetylated lipoproteins only competed for a small fraction of OxLDL binding. Both the isolated lipids and the isolated protein from OxLDL bound with high affinity to SR-BI and showed partial reciprocal competition. Monoclonal antibody EO6, an antibody against oxidized phospholipids, and 1-palmitoyl-2-(5-oxovaleroyl) phosphatidylcholine (POVPC) both competed effectively with intact OxLDL and with isolated lipids from OxLDL for SR-BI binding.Together, these results demonstrate a potential function of SR-BI, in addition to its role in selective uptake of lipids, to mediate internalization of OxLDL by macrophages and suggest a central role for oxidized phospholipids in this process.     

Scavenger receptor class B, type I-mediated uptake of various lipids into cells. Influence of the nature of the donor particle interaction with the receptor

Scavenger receptor (SR)-BI is the first molecularly defined receptor for high density lipoprotein (HDL) and it can mediate the selective uptake of cholesteryl ester into cells. To elucidate the molecular mechanisms by which SR-BI facilitates lipid uptake, we examined the connection between lipid donor particle binding and lipid uptake using kidney COS-7 cells transiently transfected with SR-BI. We systematically compared the uptake of [(3)H]cholesteryl oleoyl ether (CE) and [(14)C]sphingomyelin (SM) from apolipoprotein (apo) A-I-containing reconstituted HDL (rHDL) particles and apo-free lipid donor particles. Although both types of lipid donor could bind to SR-BI, only apo-containing lipid donors exhibited preferential delivery of CE over SM (i.e. nonstoichiometric lipid uptake). In contrast, apo-free lipid donor particles (phospholipid unilamellar vesicles, lipid emulsion particles) gave rise to stoichiometric lipid uptake due to interaction with SR-BI. This apparent whole particle uptake was not due to endocytosis, but rather fusion of the lipid components of the lipid donor with the cell plasma membrane; this process is perhaps mediated by a fusogenic motif in the extracellular domain of SR-BI. The interaction of apoA-I with SR-BI not only prevents fusion of the lipid donor with the plasma membrane but also allows the optimal selective lipid uptake. A comparison of rHDL particles containing apoA-I and apoE-3 showed that while both particles bound equally well to SR-BI, the apoA-I particle gave approximately 2-fold greater CE selective uptake. Catabolism of all major HDL lipids can occur via SR-BI with the relative selective uptake rate constants for CE, free cholesterol, triglycerides (triolein), and phosphatidylcholine being 1, 1.6, 0.7, and 0.2, respectively. It follows that a putative nonpolar channel created by SR-BI between the bound HDL particle and the cell plasma membrane is better able to accommodate the uptake of neutral lipids (e.g. cholesterol) relative to polar phospholipids.     

Reconstituted discoidal ApoE-phospholipid particles are ligands for the scavenger receptor BI. The amino-terminal 1-165 domain of ApoE suffices for receptor binding

The high density lipoprotein receptor, scavenger receptor class B type I (SR-BI), recognizes lipid-bound apolipoprotein A-I (apoA-I) and other apolipoproteins. Here, we have used large scale cultures of apoE-expressing cells to purify apoE and prepare apoE containing reconstituted discoidal 1-palmitoyl-2-oleoyl-l-phosphatidylcholine (POPC)-apoE particles. These particles have been used to examine their binding to wild-type and mutant forms of SR-BI expressed in transfected ldlA-7 cells. Specific binding to SR-BI was determined by subtracting from the total binding, nonspecific values measured using either control untransfected ldlA-7 cells or by inhibiting SR-BI-mediated binding with a high titer antireceptor-blocking antibody. POPC-apoE particles generated using apoE2, apoE3, apoE4, or the carboxyl-terminally truncated forms apoE165, apoE202, apoE229, and apoE259 all bound tightly to wild-type SR-BI with similar affinities (K(d) = 35-45 microg/ml). Binding was nearly abolished in a cell line expressing the ldlA (Q402R/Q418R) double mutant form of SR-BI that is unable to bind native high density lipoprotein but binds low density lipoprotein normally. The findings establish that apoE is a ligand for SR-BI and that the receptor binding domain is located in the amino-terminal 1-165-region of the protein. SR-BI-apoE interactions may contribute to cholesterol homeostasis in tissues and cells expressing SR-BI that are accessible to apoE-containing lipoproteins.     

Molecular characterization of the interaction between sialylated Neisseria gonorrhoeae and factor H

Human factor H (HufH), a key inhibitor of the alternative pathway of complement, binds to Neisseria gonorrhoeae and constitutes an important mechanism of human-specific complement evasion. The C-terminal domain 20 of HufH contains the binding site for sialylated gonococci. We exploited differences in amino acid sequences between human and non-binding chimpanzee fH domain 20 to create cross-species mutations to define amino acids important for binding to sialylated gonococci. We used fH/Fc fusion constructs that contained contiguous fH domains 18-20 fused to Fc fragments of murine IgG2a. The Fc region was used both as a tag for detection of each fusion molecule on the bacterial surface and as an indicator for complement-dependent killing. Arg-1203 was critical for binding to both porin (Por) B.1A and PorB.1B strains. Modeling of the R1203N human-to-chimpanzee mutation using the crystal structure of HufH19-20 as a template showed a loss of positive charge that protrudes at the C terminus of domain 20. We tested the functional importance of Arg-1203 by incubating sialylated gonococci with normal human serum, in the presence of wild-type HufH18-20/Fc or its R1203A mutant. Gonococci bound and were killed by wild-type HufH18-20/Fc but not by the R1203A mutant. A recombinant fH/Fc molecule that contained chimpanzee domain 20, humanized only at amino acid 1203 (N1203R) also bound to sialylated gonococci and restored killing. These findings provide further insights into the species specificity of gonococcal infections and proof-of-concept of a novel therapeutic approach against gonorrhea, a disease rapidly becoming resistant to conventional antibiotics.     

Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes

Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.     

Expression of a recombinant chimeric protein of human colorectal cancer antigen GA733-2 and Fc fragment of antibody using a replicating vector based on Beet curly top virus in infiltrated Nicotiana benthamiana leaves

We describe expression and characterization of recombinant human colorectal cancer antigen GA733-2 fused to Fc fragment of antibody (GA733-2-Fc) using a replicating vector based on Beet curly top virus in infiltrated Nicotiana benthamiana leaves. Recombinant GA733-2-Fc/KDEL with a molecular mass of ~68?kDa was transiently expressed. The level of expression of GA733-2-Fc with ER retention signal KDEL (GA733-2-Fc/KDEL) in the expression vector system was 0.96% of total soluble proteins. Recombinant GA733-2-Fc/KDEL was purified using an affinity chromatography. Mice immunized with recombinant GA733-2-Fc/KDEL mounted a strong GA733-2-Fc/KDEL-specific serum antibody response. Vaccination of plant-derived recombinant GA733-2-Fc/KDEL regressed tumor volumes in BALB/c mice. The population of activated-T and NK-T cells increased notably in lymph node, spleen, and tumor-infiltrating lymphocytes derived from the tumor-regressed mice. Taken together, recombinant GA733-2-Fc/KDEL expressed in plants can be used as an effective experimental immunogen for research in cancer vaccine development.     

Mutations conferring resistance to neutralization by a soluble form of the neurotrophin receptor (p75NTR) map outside of the known antigenic sites of the rabies virus glycoprotein

The neurotrophin receptor (p75NTR) serves as a receptor for rabies virus (RV). We expressed and purified a soluble chimera consisting of the p75NTR ectodomain fused to the human immunoglobulin G1 (IgG1) Fc fragment (p75-Fc). Although p75-Fc interacts with RV, the infectivity of RV did not decrease significantly when it was incubated in the presence of the soluble receptor alone. However, when it was subsequently incubated with an antihuman IgG directed against the Fc fragment of p75-Fc, the infectivity of RV was significantly lowered (>90%), whereas incubation with antihuman IgG alone had no effect. We then selected eight independent RV mutants that were not neutralized by p75-Fc and antihuman IgG (srr [soluble receptor resistant] mutants). Each mutant carried a single mutation in the glycoprotein gene leading to one amino acid substitution in the protein. A total of four different substitutions were found. Two of the mutations were located at position 318 (phenylalanine replaced by a serine or a valine residue), and two were located at position 352 (histidine replaced by a tyrosine or an arginine residue). All of the mutations prevented the interaction with p75NTR as either a soluble or a membrane-anchored form. Two mutants (F318S) and (H352R) resulted in the formation of small plaques on BSR cells, probably due to the slower maturation of the glycoprotein. Immunoprecipitation, immunofluorescence, and neutralization assays showed that the four mutated glycoproteins still interacted with representative anti-RV glycoprotein monoclonal antibodies (MAbs), indicating that p75NTR binds outside of the known RV glycoprotein antigenic sites.     

Scavenger receptor BI transfers major lipoprotein-associated phospholipids into the cells

The phospholipids of lipoproteins can be transferred to cells by an endocytosis-independent uptake pathway. We analyzed the role of scavenger receptor BI (SR-BI) for the selective cellular phospholipid import. Human monocytes rapidly acquired the pyrene (py)-labeled phospholipids sphingomyelin (SM), phosphatidylcholine, and phosphatidylethanolamine from different donors (low and high density lipoproteins (LDL, HDL), lipid vesicles). The anti-SR-BI antibody directed against the extracellular loop of the membrane protein lowered the cellular import of the phospholipids by 40-80%. The phospholipid transfer from the lipid vesicles into the monocytes was suppressed by LDL, HDL, and apoprotein AI. Transfection of BHK cells with the cDNA for human SR-BI enhanced the cellular import of the vesicle-derived py-phospholipids by 5-6-fold. In the case of the LDL donors, transfer of py-SM to the transfected cells was stimulated to a greater extent than the uptake of the other py-phospholipids. Similar differences were not observed when the vesicles and HDL were used as phospholipid donors. The concentration of LDL required for the half-maximal phospholipid import was close to the previously reported apparent dissociation constant for LDL binding to SR-BI. The low activation energy of the SR-BI-mediated py-phospholipid import indicated that the transfer occurs entirely in a hydrophobic environment. Disruption of cell membrane caveolae by cyclodextrin treatment reduced the SR-BI-catalyzed incorporation of py-SM, suggesting that intact caveolae are necessary for the phospholipid uptake. In conclusion, SR-BI mediates the selective import of the major lipoprotein-associated phospholipids into the cells, the transfer efficiency being dependent on the structure of the donor lipoprotein.     

Immunoglobulin E-binding site in Fc epsilon receptor (Fc epsilon RII/CD23) identified by homolog-scanning mutagenesis.

The IgE-binding site of the human low-affinity receptor for IgE (Fc epsilon RII/CD23) has previously been mapped to the extracellular domain between amino acid residues 160 and 287. We now have investigated which conformational epitope within this domain specifies the receptor-ligand interaction. The analysis of homolog-scanning mutants expressed in mammalian cells demonstrates that amino acid side chains that affect IgE binding are located in two discontinuous segments, between residues 165-190 and 224-256. The overall structure of the chimeric binding domains, as probed with 11 conformation-sensitive monoclonal antibodies, is generally not distorted, except by replacement of residues 165-183. In this region, disruption of binding function appears to be caused by global conformational constraints on the binding site. Substitution and deletion mutants demonstrate that six out of eight extracellular cysteines, Cys163, Cys174, Cys191, Cys259, Cys273, and Cys282, are necessary for IgE binding and are most likely involved in intramolecular disulfide bridges. We show that the Fc epsilon RII domain delineated by Cys163 and Cys282 encodes all the structural information required to form the IgE-binding site.     

Expression pattern, subcellular localization and structure--function relationship of rat Tpx-1, a spermatogenic cell adhesion molecule responsible for association with Sertoli cells

The gene for a testicular cell adhesion protein called Tpx-1, which mediates the binding of spermatogenic cells to Sertoli cells of the rat in primary culture, was previously cloned. Here the characterization of Tpx-1 is reported. Tpx-1 messenger ribonucleic acid (mRNA) became detectable in pachytene spermatocytes and continued to be present throughout development into elongated spermatids, while the amount of Tpx-1 protein seemed to increase some time after the increment of mRNA. Tpx-1 protein was also present, although less abundantly, in spermatozoa prepared from the epididymis. Tpx-1 contains a cluster of hydrophobic amino acid residues near the amino terminus and a cysteine-rich region in the carboxyl-terminal half. Tpx-1 fused with green fluorescence protein was secreted into the medium when expressed in a cultured cell line, depending on the presence of the amino-terminal hydrophobic region. Moreover, Tpx-1 was present in the medium of testicular cell primary culture. Structure-function analysis revealed that the amino-terminal 101 amino acid residues were sufficient for cell adhesion activity, whereas the carboxyl-terminal cysteine-rich region was dispensable. In conclusion, Tpx-1 is produced and secreted from spermatogenic cells at various differentiation stages, and mediates the interaction of those cells with Sertoli cells.