Human Membrane Proteins Research Articles

Expression of PEX11β Mediates Peroxisome Proliferation in the Absence of Extracellular Stimuli

Mammalian cells typically contain hundreds of peroxisomes but can increase peroxisome abundance further in response to extracellular stimuli. We report here the identification and characterization of two novel human peroxisomal membrane proteins, PEX11alpha and PEX11beta. Overexpression of the human PEX11beta gene alone was sufficient to induce peroxisome proliferation, demonstrating that proliferation can occur in the absence of extracellular stimuli and may be mediated by a single gene. Time course studies indicated that PEX11beta induces peroxisome proliferation through a multistep process involving peroxisome elongation and segregation of PEX11beta from other peroxisomal membrane proteins, followed by peroxisome division. Overexpression of PEX11alpha also induced peroxisome proliferation but at a much lower frequency than PEX11beta in our experimental system. The patterns of PEX11alpha and PEX11beta expression were examined in the rat, the animal in which peroxisome proliferation has been examined most extensively. Levels of PEX11beta mRNA were similar in all tissues examined and were unaffected by peroxisome-proliferating agents. Conversely, PEX11alpha mRNA levels varied widely among different tissues, were highest in tissues that are sensitive to peroxisome-proliferating agents, and were induced more than 10-fold in response to the peroxisome proliferators clofibrate and di(2-ethylhexyl) phthalate. Taken together, these data implicate PEX11beta in the constitutive control of peroxisome abundance and suggest that PEX11alpha may regulate peroxisome abundance in response to extracellular stimuli.

Fourier Transform Infrared Spectroscopic Studies on the Effect of Cyclic Adenosine Monophosphate on the Secondary Structure of Human Erythrocyte Membrane Proteins

In this paper, the study on the effect of cAMP on erythrocyte membrane proteins by FTIR, deconvolution and curvefitting was reported. It was found that cAMP affects the secondary structure of membrane proteins by changing random and β-turn regions to the α-helix segments. The regulation of cAMP has a best concentration region, during which cAMP has the strongest regulating function. Meanwhile, cGMP and ATP has a negative effect on membrane proteins' secondary structure comparing to cAMP.

Characterization of a 76 kDa endosomal, multispanning membrane protein that is highly conserved throughout evolution.

We report here the identification and characterization of a human 76kDa membrane protein that is found predominantly in endosomes. This protein is related to the Saccharomyces cerevisiae EMP70 gene product, a precursor protein whose 24kDa cleavage product (p24a) was found in yeast endosome-enriched membrane fractions (Singer-Krüger et al., 1993. J. Biol. Chem. 268, 14376-14386). Northern blot analysis indicated that p76 mRNA is highly expressed in human pancreas but could be detected in all tissues examined. p76 is highly conserved throughout evolution, as related proteins have also been detected in Caenorhabditis elegans and Arabidopsis thaliana. This family of proteins has a relatively divergent, hydrophilic N-terminal domain and a well-conserved, highly hydrophobic C-terminal domain which contains nine potential membrane-spanning domains. Transiently expressed, myc-tagged human p76 appears to be localized to endosomes by virtue of its apparent colocalization with transferrin receptors and some mannose 6-phosphate receptors. Furthermore, p76 adopts a type-I topology within the membrane, with its hydrophilic N-terminus facing the lumen of cytoplasmic membranes. The structural features of p76 suggest that it may function as a channel or small molecule transporter in intracellular compartments throughout phylogeny. 1998 Elsevier Science B.V.

Photochemical labeling of human erythrocyte membrane proteins with radioiodinated 4-azidosalicylic acid derivatives of G(M3), G(D3), G(M1), and FucG(M1) gangliosides.

Photoreactive gangliosides of high specific radioactivity may prove useful for studies on glycosphingolipid functions. We prepared 4-azidosalicylic acid (ASA) acylated derivatives of GM3, GD3, GM1, and FucGM1 gangliosides (gangliosides-ASA). Gangliosides-ASA were characterized by their TLC mobility, UV spectra, carbohydrate composition, and digestion with leech endoceramidase. After radioiodination to about 200 Ci/mmole gangliosides-ASA were used for photochemical labeling of human erythrocytes. Radioiodinated gangliosides-ASA were incorporated into erythrocytes in a time and concentration dependent manner, the kinetics and extent of incorporation being similar for all the gangliosides-ASA used. Radioiodinated gangliosides-ASA incorporated into erythrocytes were resistant to trypsin digestion while treatment with 1% BSA removed about 90% of the label. Incubation with cholera toxin protected radioiodinated GM1-ASA and, to a lesser extent, FucGM1-ASA but not GM3-ASA and GD3-ASA, against removal with BSA. After photolysis about 40-50% of radioactivity was firmly bound to erythrocyte lipids and proteins. The ratio of lipid- to protein-bound radioactivity ranged from 2.2:1 to 3.2:1. Photolabeled proteins were analyzed by SDS/PAGE followed by autoradiography. Band 3 was the most extensively photolabeled protein with all the radioiodinated gangliosides-ASA used. DIDS, an inhibitor of band 3 protein activity, caused reduction in photolabeling of this protein by about 20%.

Interaction of Human Plasma Membrane Proteins and Oligodeoxynucleotides

Two oligodeoxynucleotide (oligodN) binding proteins of 100–110 kDa on plasma membranes of human cell lines were recently identified by us. These two proteins seemed to play a role in oligodN uptake. In this study, the impact of the chain length and the sequence of the oligodN on the interaction with those two proteins was investigated. Chain length of oligodN was an important determinant, but not the sole determinant for the interaction. Binding affinity of oligodNs was determined predominantly by base composition, where pyrimidine bases but not purine bases were required in the sequence to retain high affinity. The binding kinetics of the homopolymers of deoxycytidine (dC 21) and deoxythymidine (dT 21) suggests that the proteins may have different binding sites, with one site preferring thymine bases and the other cytosine bases. Moreover, some additional plasma membrane proteins were identified, with an apparent molecular mass ranging from 40 to 58 kDa, which could bind thymine bases but not cytosine bases.

Cloning of a Novel Human Putative Type Ia Integral Membrane Protein Mapping to 21q22.3

The distal part of human chromosome 21q22.3 is exceptionally gene rich and contains several loci that have been linked to hereditary disorders. In the course of constructing an extensive transcript map for chromosome 21, we have isolated numerous coding segments in 21q22.3 that represent potential candidate genes in this region. Following this approach, we have cloned a novel single-copy gene (C21orf3) (HGMW-approved symbolC21orf1) expressed as a unique 2.69-kb mRNA in a wide range of tissues. We have precisely mappedC21orf3by fiber FISH distal to marker D21S171. TheC21orf3gene encodes a predicted protein of 180 residues that does not share any sequence homology with other known proteins. C21orf3 harbors predicted structural features of a type Ia integral membrane protein and contains a tetrapeptide motif (YXRF) observed in several cell surface proteins involved in signal transduction. Although the function ofC21orf3is still unknown, this novel gene may play an important role in a cell trafficking mechanism.

Antigenic Homology of HIV-1 GP41 and Human Platelet Glycoprotein GPIIIa (Integrin β3)

Fifty-eight of 89 serum samples (65.17%) from HIV-1-infected individuals at various disease stages contain antibodies that react with a platelet peptide located in the cytoplasmic domain of integrin beta3, glycoprotein GPIIIa (aa749-761; sequence DRKEFAKFEEERA). Rabbit polyclonal antibodies raised against the synthetic platelet peptide also react with the structurally homologous HIV-1 gp41-derived peptide (EKNEQELLELDKW(A)) and bind to a Western blot band with molecular weight corresponding to HIV-1 gp41. These findings point to molecular mimicry between HIV-1 and a human membrane protein found in platelets and other cells that could be of pathologic consequence.

Solubilization and biochemical characterization of the human myometrial corticotrophin-releasing hormone receptor

We have solubilized an active form of the myometrial corticotrophin-releasing hormone (CRH) receptor using 1% w/v digitonin. The solubilized receptor retains its capacity for high-affinity binding as demonstrated by Scatchard analysis, although there was a shift in dissociation constant ( K d) from 83.6±15–195±35 pM for the membrane-bound and soluble receptor respectively. There was no difference in the maximum binding site concentrations ( B max) of 13±5 and 21.5±6 fmol/mg protein for the membrane-bound and soluble receptor respectively. Sauvagine unlike CRH had no effect on radiolabeled CRH binding which suggests that the CRH-R2 receptor is not present in the myometrium. The solubilized receptor did not retain guanine-nucleotide sensitivity. The isoelectric focusing (IEF) profile of the human myometrial CRH receptors was significantly different from that of the rat cerebral cortex. Furthermore, solubilization of human myometrial membrane proteins followed by gel filtration and SDS-PAGE revealed a specifically labeled protein with an apparent molecular weight of 42 000–47 000 kDa. Our results suggest that during solubilization the human myometrial CRH receptor is dissociated from the guanine nucleotide-binding protein (G s) and that high affinity binding for soluble CRH receptors is not dependent on the coupling of a guanine nucleotide-binding protein.

Five monoclonal antibodies against glycophorin A of human erythrocyte recognize glycoprotein of bovine erythrocyte.

To study heterophile blood antigens on erythrocytes between human and experimental or domestic animals, we have produced 295 monoclonal antibodies (MAbs) to human erythrocyte membrane protein. According to the affinity, reactivity, and titre of the MAbs, we selected 40 clones to study the heterophile blood antigens between human and bovine, chicken, guinea pig, horse, rabbit, sheep, and swine. Five MAbs commonly reacted with human type A, type B, and type O erythrocytes and reacted with bovine erythrocytes as well but did not react with erythrocytes from other species. Other MAbs did not react with erythrocytes from all the tested animals. These five MAbs reacted with the same erythrocyte membrane protein, 90 KD glycophorin A (GPA) of human or 200 KD major glycoprotein and other two components of bovine by immunoblotting and GPA competitive inhibition assay. Furthermore, by enzyme treatment and monosaccharide competitive inhibition assay, it was confirmed that these five MAbs recognized antigen epitope of glycosylation free amino acid portion but not glycosylation portion of GPA of erythrocyte membrane.

Identity of a conserved motif in phospholipid scramblase that is required for Ca2+-accelerated transbilayer movement of membrane phospholipids.

Accelerated transbilayer movement of plasma membrane phospholipids (PL) upon elevation of Ca2+ in the cytosol plays a central role in the initiation of plasma clotting and in phagocytic clearance of injured or apoptotic cells. We recently identified a human erythrocyte membrane protein that induces rapid transbilayer movement of PL at elevated Ca2+. We also presented evidence that this PL scramblase is expressed in a variety of other cells and tissues where transbilayer movement of plasma membrane PL is promoted by intracellular Ca2+ [Zhou, Q., et al. (1997) J. Biol. Chem. 272, 18240-18244]. We have now cloned murine PL scramblase for comparison with the human polypeptide. Both human and murine PL scramblase are acidic proteins (pI = 4.9) with a predicted inside-outside (type 2) transmembrane segment at the carboxyl-terminus. Whereas human PL scramblase (318 AA) terminates in a short exoplasmic tail, murine PL scramblase (307 AA) terminates in the predicted membrane-inserted segment. The aligned polypeptide sequences reveal 65% overall identity, including near identity through 12 residues of an apparent Ca2+ binding motif (D[A/S]DNFGIQFPLD) spanning codons 273-284 (human) and 271-282 (murine), respectively. This conserved sequence in the cytoplasmic domain of PL scramblase shows similarity to Ca2+-binding loop motifs previously identified in known EF hand structures. Recombinant murine and human PL scramblase were each expressed in Escherichia coli and incorporated into proteoliposomes. Measurement of transbilayer movement of NBD-labeled PL confirmed that both proteins catalyzed Ca2+-dependent PL flip-flop similar to that observed for the action of Ca2+ at the cytoplasmic face of plasma membranes. Mutation of residues within the putative EF hand loop of human PL scramblase resulted in loss of its PL mobilizing function, suggesting that these residues directly participate in the Ca2+-induced active conformation of the polypeptide.

Expression analysis of human Rhesus blood group antigens by gene transduction into erythroid and non-erythroid cells.

Rh blood group antigens are associated with non-glycosylated human erythrocyte membrane proteins encoded by two closely related genes, RHCE and RHD, and with a glycoprotein, a critical co-expressing factor encoded by the RH50 gene. The sequence analysis of RHCE transcripts has revealed that RhE/e and C/c serological phenotypes are associated with a nucleotide substitution in exon 5 and six substitutions in exons 1 and 2 of RHCE gene, respectively. Smythe et al. have shown that the full length transcript of RhcE gene expressed c and E antigens and the transcript of RhD gene expressed D and G antigens, using retroviral-mediated gene transduction into K562 cells. We performed an epitope analysis of Rh antigen by constructing retroviral gene coding six RH cDNAs, which contain RhcE, ce, CE and D cDNAs, and CE-D, D-CE chimera cDNAs. The cDNAs were introduced into KU812E cells and the expressed antigens were analyzed by flow cytometry. These studies revealed that the C/c and E/e associated substitutions actually participated in respective polymorphic epitopes. However, the C antigen was not detected on the KU812E cells introduced with CE cDNA, despite E antigen being expressed. The study with the chimera gene between CE and D cDNAs also indicated that the Rh epitopes were not constructed with short polymorphic exofacial peptide loops only but also with other peptide fragments and membrane components. Co-expression studies of Rh50 and RhD or cE gene in non-erythroid cells, 293, and expression studies of Rh50 in another erythroid cell, HEL, did not show any Rh antigens on the transduced cells, despite the Northern blot study showing both transcripts in the cells. It was suggested that at least a second co-expressing factor was needed to express RhCE or D antigens on the plasma membrane.

Cloning and tissue expression of two putative steroid membrane receptors.

We have cloned two human putative steroid binding membrane proteins, termed Hpr6.6 and Dg6. Hpr6.6 is the human homolog of a previously cloned porcine progesterone binding protein. Both proteins contain a putative transmembrane domain and a highly conserved stretch of 58 amino acids. Hpr6.6 mRNA is expressed predominantly in liver and kidney, whereas Dg6 mRNA is preferentially expressed in placenta. Hpr6.6 is located on the X chromosome and dg6 on chromosome 4. The two proteins are the first putative steroid membrane receptors cloned from man.

Comparison of the abilities of proteins from Bartonella bacilliformis and Bartonella henselae to deform red cell membranes and to bind to red cell ghost proteins

Infections in humans by Bartonella bacilliformis, but not Bartonella henselae, are characterized by invasion of red cells. Supernatants of culture medium from B. bacilliformis and B. henselae each contain a protein which causes invagination of membranes of human red cells and formation of intracellular vacuoles. These two proteins are very similar in molecular mass, heat stability and mechanism of action. B. henselae does not bind to human red cells, but human red cell ghost membrane proteins were recognized by both bacteria, five by B. bacilliformis and the same five, and one additional protein by B. henselae. Two of these proteins had molecular masses consistent with actin and spectrin. Actin binds to five electroblotted outer membrane proteins from B. henselae and four of these proteins are retained on an actin-Sepharose column.

Identification of Endothelial Cell Membrane Proteins that Bind Anti-DNA Antibodies from Patients with Systemic Lupus Erythematosus by Direct or Indirect Mechanisms

A subgroup of murine monoclonal anti-DNA antibodies bind to vascular endothelial cells either directly as a result of cross-reactivity, or indirectly through immunoglobulin-bound DNA and DNA-binding proteins on the endothelial cell membrane. To determine whether these mechanisms apply in human systemic lupus erythematosus (SLE), and to identify endothelial cell membrane protein(s) that bind human anti-DNA antibodies, we examined, by Western blotting, the binding of human polyclonal anti-DNA antibodies (PoAb) isolated from eight patients with SLE to human umbilical vein endothelial cell membrane proteins. PoAbs bind to endothelial membrane proteins with Mr84,000 and 46,000, which correspond to the DNA-binding proteins previously reported. Such binding is diminished after removal of DNA by DNase treatment. In addition, PoAbs bind to membrane proteins with Mr180,000, 110,000, 68,000, 44,000, and 35,000–30,000. Such binding is unaffected by alterations in DNA concentration. Anti-dsDNA and anti-ssDNA PoAbs from individual patients exhibit identical binding patterns, as are PoAbs isolated during active disease or remission. The results show that human anti-DNA antibodies can bind to endothelial cells both indirectly via immunoglobulin-bound DNA, and directly due to cross-reactivity. These mechanisms of cellular binding by anti-DNA antibodies may depict patho-genetic steps in human SLE.

Identification of human liver carboxylesterase as one of the proteins involved in Plasmodium falciparum malaria sporozoite invasion in primary cultures of human hepatocytes

Background/Aims: In a previous study, we have demonstrated that primary human hepatocytes in culture are susceptible for Plasmodium falciparum sporozoite invasion and for development of parasites into exoerythrocytic forms. In a separate study we demonstrated the involvement of two human liver plasma membrane proteins (55 kD and 20 kD) in the invasion of P. falciparum sporozoites in vitro. In this study we have unravelled the nature of the 55 kD protein. Methods: For the identification of this protein, a 53–58 kD membrane protein fraction from human liver was isolated, radioactively labelled, incubated with sporozoites and cross-linked. After reduction of the cross-linker, the released proteins were mixed with unlabelled 53–58 kD protein fraction and separated on two-dimensional SDS-PAGE. Autoradiography showed a single spot corresponding to a protein of 55 kD and pI of 5.7–5.8. Results: Amino acid sequencing revealed the 55 kD protein as carboxylesterase. The biological activity of purified human liver carboxylesterase and of an anti-serum against carboxylesterase on sporozoite invasion in vitro was evaluated. Human carboxylesterase as well as a rabbit antiserum against carboxylesterase inhibited the invasion of P. falciparum sporozoites into primary human hepatocytes in culture. A number of carboxylesterase cDNA clones were isolated from a human liver cDNA library. Sequence analysis revealed two different iso-types. Immunoaffinity purified recombinant human carboxylesterase was shown also to inhibit the invasion of sporozoites into primary human hepatocytes. Immunocytochemical analysis of the localisation of carboxylesterase in primary cultures of human hepatocytes using specific antibodies, showed its presence inside the hepatocytes and on the membrane. Conclusions: Carboxylesterase plays a role in the invasion process of P. falciparum sporozoites into human hepatocytes in vitro. The implications of these findings are further discussed.

Cloning of a human multispanning membrane protein cDNA: evidence for a new protein family

We report the cloning of a human cDNA encoding a protein of calculated 68.8 kDa molecular mass, named hMP70. The deduced protein sequence shows a large N-terminal hydrophilic part and a C-terminal part with nine putative hydrophobic regions characteristic of integral transmembrane domains. Computer searches with sequence databases revealed homologies with three complete yeast proteins and with at least 19 human, 10 plant and one nematode short unidentified protein sequences translated from Expressed Sequence Tags (ESTs). Remarkably, this hMP70 protein retains between 27 and 31% overall sequence identity with the yeast proteins. We propose that hMP70 and related genes have evolved from a common ancestral gene and form a new multispanning membrane protein family which we call the MP70 protein family. Gene expression of hMP70 appears to be ubiquitous, as the mRNA is detectable in all human tissues analysed so far, as shown by Northern blot analysis. Furthermore, a protein of about 70 kDa is detectable in different mammalian cell lines, as shown by immunoblot analysis. From its widespread expression and conservation from yeast, plants to mammals, it is likely that hMP70 has a fundamental biological function in the cell.

R-023. Characterization of human sperm membrane proteins

R-023. Characterization of human sperm membrane proteins

Domain-specific Interactions of Human HP1-type Chromodomain Proteins and Inner Nuclear Membrane Protein LBR

HP1-type chromodomain proteins self-associate as well as interact with the inner nuclear membrane protein LBR (lamin B receptor) and transcriptional coactivators TIF1alpha and TIF1beta. The domains of these proteins that mediate their various interactions have not been entirely defined. HP1-type proteins are predicted by hydrophobic cluster analysis to consist of two homologous but distinct globular domains, corresponding to the chromodomain and chromo shadow domain, separated by a hinge region. We show here that the chromo shadow domain mediates the self-associations of HP1-type proteins and is also necessary for binding to LBR both in vitro and in the yeast two-hybrid assay. Hydrophobic cluster analysis also predicts that the nucleoplasmic amino-terminal portion of LBR contains two globular domains separated by a hinge region. The interactions of the LBR domains with an HP1-type protein were also analyzed by the yeast two-hybrid and in vitro binding assays, which showed that a portion of the second globular domain is necessary for binding. The modular domain organization of HP1-type proteins and LBR can explain some of the diverse protein-protein interactions at the chromatin-lamina-membrane interface of the nuclear envelope.

Effector Cell Protease Receptor-1, a Platelet Activation-dependent Membrane Protein, Regulates Prothrombinase-catalyzed Thrombin Generation

At sites of vascular injury thrombin is generated via prothrombinase, a stoichiometric (1:1), Ca2+-dependent, and membrane-bound complex consisting of the nonenzymatic cofactor factor Va and the serine protease factor Xa. While the importance of anionic platelet membrane phospholipids in regulating thrombin generation is well recognized, the identification of regulatory protein receptors has eluded investigators. This study reports the first description of a human platelet membrane protein that regulates prothrombinase complex assembly and function. Direct platelet-protein binding studies indicated that, although required, platelet-bound factor Va alone is insufficient to mediate factor Xa binding, and that factor Va and factor Xa bind to discrete sites on activated platelets for which expression is independently regulated as a function of the agonist concentration. When specific monoclonal antibodies against effector cell protease receptor-1 (EPR-1, a 65-kDa membrane receptor for factor Xa) were used in Western blotting, immunohistochemical staining, and/or flow cytometric analyses, activated platelets and their precursors, megakaryocytes, were shown to express EPR-1. These results were confirmed by reverse transcription-polymerase chain reaction of mRNA extracted from megakaryocyte-like cell lines. Additional flow cytometric studies demonstrated that a platelet-bound factor Va/factor Xa complex precluded binding of the anti-EPR-1 antibody, B6, to activated platelets by approximately 50%. Likewise, the anti-EPR-1 antibody was shown to inhibit prothrombinase-catalyzed thrombin generation on activated platelets in a dose- and platelet donor-dependent manner, indicating that platelet-expressed EPR-1 mediates factor Xa assembly into the prothrombinase complex. These collective data indicate that both EPR-1 and membrane-bound factor Va are required to mediate factor Xa binding to the activated platelet to form a functional prothrombinase complex.

CDNA Cloning, Genomic Structure, and Chromosome Mapping of the Human Epithelial Membrane Protein CL-20 Gene (EMP1), a Member of the PMP22 Family

CL-20is a novel gene encoding a protein that is structurally related to but distinct from the peripheral myelin protein PMP22. Like PMP22, CL-20 is likely to play important roles in the regulation of cell proliferation, differentiation, and cell death. In this study, we describe the cloning and sequencing of a cDNA encoding the human homologue of CL-20 and characterize the genomic structure of this gene. ThehCL-20gene (HGMW-approved symbol EMP1) encodes a protein of 157 amino acids that exhibits 76% identity to the rabbit CL-20 and to the rat EMP-1, which have been described recently, and 39% identity to human PMP22. CL-20 contains four hydrophobic domains, suggesting that it is an integral membrane protein. In particular the second hydrophobic domain encoded within the fourth exon is highly conserved among CL-20, EMP-1, and PMP22, suggesting a functional role for this region. CL-20 mRNA is abundant in squamous-differentiated bronchial epithelial cells; however, low levels of CL-20 mRNA can be detected in several human tissues by Northern analysis. Retinoic acid, which inhibits squamous differentiation, represses CL-20 expression in normal human bronchial epithelial cells. The genomic structure of thehCL-20gene was analyzed using a P1 vector containing this gene. ThehCL-20gene contains five exons about 0.2, 0.12, 0.1, 0.14, and 2.2 kb and four introns about 15, 1.9, 0.1, and 0.7 kb. We have mapped thehCL-20gene to chromosome 12p12 by fluorescencein situhybridization.