Kilodalton Protein Research Articles

Transportin: Nuclear Transport Receptor of a Novel Nuclear Protein Import Pathway

Many nuclear proteins are imported into the cell nucleus by the “classical” nuclear localization signal (NLS)-mediated import pathway. In this pathway, a sequence rich in basic residues in the protein interacts with a heterodimeric complex termed importin and this, along with the GTPase Ran, mediates nuclear import of the NLS-bearing protein. The heterogeneous nuclear ribonucleoprotein (hnRNP) A1 protein contains a novel nuclear localization sequence, termed M9, that does not contain any clusters of basic residues. Very recently, we showed that M9 directs import into the nucleus by a novel protein import pathway distinct from the classical NLS pathway. A 90-kilodalton protein termed transportin was identified as a protein that specifically interacts with wild-type M9 but not transport-defective M9 mutants. Transportin and an ATP-regenerating system were found to be necessary and sufficient for import of M9-containing proteins in anin vitroimport assay. In this report, we provide additional evidence that transportin can interact directly with M9-containing proteins and also show that it can mediate import of full-length hnRNP A1. In addition, Ran, or a Ran-binding protein, is identified as a second protein component of this novel nuclear import pathway. Transportin relatives fromSaccharomyces cerevisiaewhich likely serve as additional nuclear transport receptors are described.

Purification of milligram quantities of human leptin from recombinant E. coli.

Leptin, the product of the obese (ob) gene, is a 16 kilodalton protein secreted from adipose tissue. Restoration of leptin to obese ob/ob mice leads to normalization of body weight. The effect of leptin in larger animals has not been explored, in part because of limited supplies of leptin. To date, the potency and yield of recombinant leptin purified from a variety of eukaryotic sources or from E. coli has been highly variable. While purification of leptin from E. coli inclusion bodies has afforded the greatest yield of protein, its potency is at least an order of magnitude lower than that of leptin secreted from E. coli or eukaryotic cells. The mechanistic basis of this difference in potency is not clear at present. The ability to purify significant quantities of highly active leptin will be crucial for the evaluation of leptin structure, as well as its function in additional animal models of obesity. We now report a facile protocol for the preparation of recombinant leptin using an E. coli expression system. 75-85 milligrams of leptin with a purity of greater than 97 % was prepared from a liter of recombinant E. coil. The procedure can be performed in less than 48 h and requires no chromatography. Intraperitoneal injection of 0.1 mg/kg renatured leptin into ob/ob mice results in a significant reduction in food consumption. The potency of this material is similar to the most potent recombinant leptin described to date. The ability to rapidly prepare large quantities of high specific activity material will hasten the definition of leptin's role in non-rodent models of obesity.

The relative abundance of a salivary protein, bSP30, is correlated with susceptibility to bloat in cattle herds selected for high or low bloat susceptibility

Pasture bloat is a serious economic and animal welfare problem in cattle grazed on legumes in New Zealand. Analysis of salivary proteins from dairy cattle in herds bred for either low or high susceptibility to bloat has resulted in the identification of a 30 kilodalton protein, which we term bSP30, whose relative abundance is negatively correlated with bloat score (r = -0.40 +/- 0.12). From 74 animals sampled, relative abundance of bSP30 was 66 +/- 15% higher in the low-susceptibility herd than in the high-susceptibility herd. Relative abundance of bSP30 also varied significantly within individuals, according to feeding or time of day, and from day to day. A sequence homology search of 38 amino acids derived from three tryptic fragments of the protein suggests that the amino acid sequence of bSP30 has not been described previously. Amino acid analysis indicates that bSP30 is not a member of the proline-rich family of salivary proteins. The function of bSP30 is unknown but it is conceivable that it plays a role in the aetiology of bloat.

Hyperthermia induces heat shock protein expression and protection against cerulein-induced pancreatitis in rats

BACKGROUND & AIMS: Heat shock proteins (HSPs) are part of the cellular stress response machinery. HSPs are expressed in the pancreas, but their protective potential has not been thoroughly investigated. The aim of this study was to characterize the pancreatic heat shock response both in vitro and in vivo and to study whether this response has protective effects. METHODS: For in vitro experiments, rat pancreatic acini were exposed to heat and protein expression was analyzed through 35S-methionine labeling or Western blots. In vivo, cerulein pancreatitis was induced after whole body hyperthermia (42 degrees C). RESULTS: After thermal stress (42 degrees C), acini increasingly expressed five proteins of 92, 72, 59, 58, and 30 kilodaltons, with HSP. 70 the most strongly induced 72-kilodalton protein. In vivo, increased expression of four isoforms of pancreatic HSP-70 was found. Isoform-specific antibodies identified the inducible and constitutively expressed (HSC-70) isoform of HSP-70. HSP-60 was also weakly induced after hyperthermia. Concomitantly, cerulein-induced pancreatic organ damage was greatly reduced after whole-body hyperthermia. The time course and strength of HSP induction correlated well with the time course and degree of protection against cerulein- induced pancreatitis. CONCLUSIONS: A causal relation between hyperthermia-induced HSP expression and organ protection seems possible. (Gastroenterology 1996 Nov;111(5):1333-42)

Evidence for cell-surface association between fusin and the CD4-gp120 complex in human cell lines.

Accessory cell-surface molecules involved in the entry of human immunodeficiency virus-type 1 into cells have recently been identified and shown to belong to the family of chemokine receptors. Treatment of human cell lines with soluble monomeric gp120 at 37 degrees C induced an association between the surface CD4-gp120 complex and a 45-kilodalton protein, which can be down-modulated by the phorbol ester phorbol 12-myristate 13-acetate. The three proteins were coprecipitated from the cell membranes with antibodies to CD4 or to gp120. The 45-kilodalton protein comigrated with fusin on sodium dodecyl sulfate gels and reacted with rabbit antisera to fusin in protein immunoblots. No 45-kilodalton protein could be coprecipitated from similarly treated nonhuman cells. However, infection of 3T3.CD4.401 cells with vaccinia-fusin recombinant virus (vCBYF1), followed by gp120 treatment, resulted in coprecipitation of fusin and CD4.401 molecules from their membranes. Together these data provide evidence for physical association between fusin and the CD4-gp120 complex on cell membranes.

An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.

The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.

The Candidates Speak

An Adenovirus Mutant That Replicates Selectively in p53- Deficient Human Tumor Cells The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.

Enhancement by melatonin of GTP exchange and ADP ribosylation reactions

The pineal hormone melatonin modulates constitutive protein secretion from murine melanoma M2R cells in vitro, in a cholera-toxin (CTX)-sensitive process, without effecting major changes in cAMP. The effects of melatonin on GTP binding proteins and putative CTX substrates in these cells were investigated. Melatonin enhanced GTPγ 35S binding and the incorporation of 32P-P 3-(4-azidoanilido)-P 1-5′-guanosine triphosphate (Az- 32P-GTP) into 94, 40 and 28 kilodalton proteins. Similar changes were induced by CTX treatment. In addition, melatonin enhanced ADP ribosylation of several proteins, among them 94 and 40 kilodalton bands, apparently at arginyl residues. CTX catalyzed the ADP ribosylation of 45 and 40 (both recognized by antibodies specific to the C-terminal peptide of the Gsα subunit) and 94 kilodalton proteins and attenuated melatonin's effect. The melatonin-mediated ADP ribosylation reactions were attenuated by nicotinamide which inhibits mono(ADP ribosyl)transferases and poly(ADP-ribose)synthetase, but not by 3-amino benzamide, a specific inhibitor of poly(ADP-ribose)synthetase. Nicotinamide but not 3-amino benzamide prevented the enhancement by melatonin of GTPγ 35S binding. These results indicate that melatonin enhances protein ADP ribosylation and consequently GTP exchange in a number of CTX-sensitive G proteins. They demonstrate a novel route for concerted activation of multiple GTP binding proteins by a single hormone.

Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K.

The E1B 19-kilodalton protein (19K protein) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2 (E. White, Genes Dev. 10:1-15, 1996). To obtain a better understanding of the biochemical mechanism by which the E1B 19K protein regulates apoptosis, proteins that interact with 19K have been identified; one of these is Bax (J. Han, P. Sabbatini, D. Perez, L. Rao, D. Mohda, and E. White, Genes Dev. 10:461-477, 1996), and another is Bak (S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J.-C. Martinou, and R. Brown, Nature (London) 374:731-733, 1995). Bax and Bak are Bcl-2 family members which contain Bcl-2 homology regions 1, 2, and 3 (BH1, BH2, and BH3), which interact with E1B 19K and Bcl-2 and promote apoptosis. Like Bax and Bak, Nbk was cloned from a yeast two-hybrid screen for proteins that interact with E1B 19K. Nbk contained BH3 but not BH1 or BH2. It also interacted with Bcl-2 but not with Bax. Both Bcl-2 and E1B 19K interacted with Nbk in vitro, and this interaction was highly specific. In vivo, the Nbk and E1B 19K proteins may colocalize with cytoplasmic and nuclear membranes. Nbk expression functionally antagonized 19K-mediated inhibition of apoptotic cell death and completely prevented transformation by E1A and E1B 19K. Nbk was sufficient for induction of apoptosis in the presence of mutant p53 and thus low levels of Bax, suggesting that Nbk functions independently of Bax to induce apoptosis. Nbk may therefore represent a novel death regulator which contains only a BH3 that interacts with and antagonizes apoptosis inhibitors such as the E1B 19K protein.

Diflubenzuron stimulates phosphorylation of a 39 kDa integumental protein from newly molted American cockroach ( Periplaneta americana)

We identified a 39 kilodalton (kDa) protein from newly molted American cockroaches ( Periplaneta americana), isolated from a 100 000 g precipitate of a 1000 g supernatant from an integument homogenate, which exhibited increased phosphorylation following diflubenzuron (DFB) treatment. This 39 kDa phosphoprotein was partially purified from an intracellular membrane vesicle containing fractions obtained by discontinuous sucrose density centrifugation. Both the interfaces of the 30/40% and 40/50% sucrose density layers were found to be enriched in the 39 kDa phosphoprotein. Treatments with various chemicals such as the ionophores valinomycin and A23187, the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), and the vacuolar H +-ATPase inhibitor N-ethylmaleimide (NEM), like DFB, were also found to stimulate phosphorylation of the 39 kDa protein. These results suggest that by disrupting the normal pH gradient occurring in certain acidic vacuolar-type membrane vesicles, phosphorylation of the 39 kDa protein will be increased. In addition, we found that by decreasing the external pH to 5.0 from about neutral, it was possible to stimulate the phosphorylation activity of this particular protein, and thus simulate the action of DFB. We concluded that DFB's action is very probably related to its ability to disrupt the normal ionic balance of these vacuolar-type vesicles, leading to eventual disruption of the proton gradient within the vesicles.

Effects of CaCN2treatment of seeds of Inubie (Echinochloa oryzicola) on synthesis of protein and RNA during germination

Abstract Protein and RNA induction during the germination of Inubie (Echinochloa oryzicola) seeds pretreated with CaCN2 was studied. It was observed that the germination of Echinochloa oryzicola seeds was delayed after pretreatment with a high concentration of CaCN2 for 18 h. A considerable difference in the protein patterns was detected in the SDS-PAGE gel between the control (H2O) and CaCN2 treatment, particularly a 48 kilodalton (kD) protein band disappeared in CaCN2 treatment. At the tested three levels of CaCN2, the induction of this 48 kD protein was apparently inhibited during the germination process even when the duration of the pretreatment was as short as 2 h. When imbibed with water, the amount of the 48 kD protein increased rapidly within 1 h from the trace level in the dry seeds, and reached the maximum level after about 1–2 d. The results showed that the CaCN2 treatment also decreased the total RNA level in the germinating seeds. The relation of this protein induction and RNA decline with th...

Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells.

The localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B proteins was determined in the absence of other adenovirus proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B protein colocalized in the nucleus with the E4 protein. A complex of the E4 and E1B proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 protein failed to direct the E1B protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 protein to direct the nuclear localization of the E1B protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with protein synthesis inhibited, a portion of the E4 protein present in the REF-52 nuclei migrated to the HeLa nuclei. Simultaneously, the previously cytoplasmic E1B protein colocalized with the E4 protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 proteins of adenovirus.

Translation of a testis-specific Cu/Zn superoxide dismutase (SOD-1) mRNA is regulated by a 65-kilodalton protein which binds to its 5' untranslated region.

Mouse testes contain two distinct superoxide dismutase (SOD-1) transcripts which differ by 114 nucleotides in their 5' untranslated regions (UTRs) (W. Gu, C. Morales, and N. B. Hecht, J. Biol. Chem. 270:236-243, 1995). The shorter SOD-1 mRNA, a somatic type SOD-I mRNA (SSOD-1), is ubiquitously expressed in all somatic tissues as well as in testes. The larger SOD-1 mRNA, a testis-specific SOD-1 mRNA (TSOD-1), derived from an alternative upstream start site, is transcribed solely in postmeiotic germ cells and is translationally regulated during spermiogenesis. Since the two mRNAs have identical nucleotides except that TSOD-1 has an additional sequence at its 5' terminus, we have proposed that the extra 5' UTR sequence may be involved in the translational control of the TSOD-1 mRNA during spermiogenesis. Here we show that, when assayed in a cell-free system, TSOD-1 is translated only slightly less efficiently than SSOD-1. RNA gel retardation and UV cross-linking assays reveal that a testicular cytoplasmic protein (Cu/Zn superoxide dismutase RNA-binding protein [SOD-RBP]) of about 65 kDa specifically binds to the extended 5' UTR of TSOD-1. After purification of SOD-RBP by RNA affinity chromatography, we demonstrate that SOD-RBP can repress the in vitro translation of TSOD-1 mRNA but not SSOD-1 mRNA or cotranslated luciferase mRNA. We conclude that SOD-RBP serves as a repressor in the translation of TSOD-1 mRNA during spermiogenesis and thereby fine-tunes the level of Cu/Zn superoxide dismutase produced in maturing germ cells.

Selective immunomodulation: utilization of CD29/VLA molecules.

Accumulating evidence suggests that the VLA/CD29 molecule plays an important role in T-cell costimulation, and CD4+CD29/VLA+ memory T cells play a key role in induction of CD8 killer effector T cells which are considered to be a major population involved in graft rejection. To target limited elements of the T-lymphocyte population, we have described the preparation of a bispecific antibody-toxin conjugate designed to target CD4+CD29+ memory T cells. We also showed that the solid-phase crosslinking of VLA-4 by the antibody against this molecule or by its ligand, the CS-1 region of fibronectin, stimulates tyrosine phosphorylation of 140, 120-105, 80-70, 60-55, 50 and 45 kilodalton proteins. In addition, we identified the pp140 protein as PLC gamma, pp120 protein as pp125FAK, pp70 and pp50 proteins as paxillin, and pp60-55 proteins as pp59fyn and pp56lck, and pp45 as MAP kinase, respectively. Moreover, we demonstrated that pp125FAK is directly associated with paxillin. The paxillin binding domain of pp125FAK is homologous to the paxillin binding domain of vinculin. Mutations in the conserved amino acid residues between pp125FAK and vinculin result in the loss of paxillin-binding activity. Because VLA/CD29 is preferentially expressed on CD4 memory T cells, the above described system will be used to develop a novel drug design for providing selective immunosuppression useful for organ transplantation.

Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics

BACKGROUND & AIMS: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. METHODS: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. RESULTS: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively, The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. CONCLUSIONS: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease. (Gastroenterology 1996 Jul;111(1):206-16)

A 60-kilodalton protein in rat hepatoma cells overexpressing insulin receptor was tyrosine phosphorylated and associated with Syp, phophatidylinositol 3-kinase, and Grb2 in an insulin-dependent manner

A 60-kilodalton protein in rat hepatoma cells overexpressing insulin receptor was tyrosine phosphorylated and associated with Syp, phophatidylinositol 3-kinase, and Grb2 in an insulin-dependent manner

A 60-kilodalton protein in rat hepatoma cells overexpressing insulin receptor was tyrosine phosphorylated and associated with Syp, phophatidylinositol 3-kinase, and Grb2 in an insulin-dependent manner.

Tyrosine phosphorylation of cellular proteins is an early and key step after activation of the insulin receptor kinase (IRK). The study of the properties of these proteins should contribute to our understanding of insulin action. In rat hepatoma cells overexpressing human insulin receptors (HTC-IR), insulin treatment resulted in rapid tyrosine phosphorylation of proteins of 180, 94, 68, and 60 kDa. When lysates from insulin-treated cells were immunoprecipitated with anti-Syp antibody, subsequent immunoblotting identified p65 and p68, which reacted with anti-Syp, and p6O and p68, which reacted with antiphosphotyrosine antibody. Thus, insulin treatment yielded tyrosine phosphorylation of both Syp and a Syp-associated p6O molecule. When lysates from insulin-treated cells were adsorbed with a glutathione S-transferase (GST)-Syp-Src homology-2 (SH2) fusion protein, tyrosine- phosphorylated p6O was sequestered. After subjecting lysates to SDS-PAGE, the GST-SypSH2 fusion protein was found to bind to p18O, p94, and p6O. Thus, Syp associates directly with a 60-kDa IRK substrate via its SH2 domains. Syp-associated p6O differed from the 60- to 62-kDa proteins, associating with ras guanosine triphosphatase-activating protein, which also underwent modest tyrosine phosphorylation in response to insulin. Preadsorption of cell lystates with antibody against the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase substantially reduced the amount of p60 subsequently immunoprecipitated by anti-Syp. Thus, p60 associates with both Syp and p85. The amount of tyrosine-phosphorylated p60 exceeded that of p180 in anti-Syp immunoprecipitates, whereas their proportion was comparable in anti-p85 immunoprecipitates. Grb2 was also observed in the anti-Syp immunoprecipitates. When lysates from insulin-treated cells were adsorbed with GST-p85SH2 domains or GST-Grb2, the subsequent eluates contained tyrosine-phosphorylated p60, as determined by immunoblotting with antiphosphotyrosine. Membrane binding assays using GST fusion proteins showed that these associations were direct. Studies in rat liver, muscle, and adipose tissue identified insulin-dependent association of Syp, Grb2, and p85 with tyrosine-phosphorylated p60 in adipose tissue only. We conclude that insulin treatment of HTC-IR cells and rat adipose tissue results in the tyrosine phosphorylation of p60, which might participate in the recruitment of downstream effectors involved in insulin signal transduction.

Testosterone regulates tissue-specific changes in the binding of a 47- kilodalton protein to a highly conserved sequence in the 3' untranslated region of epidermal growth factor messenger ribonucleic acid

Testosterone regulates tissue-specific changes in the binding of a 47- kilodalton protein to a highly conserved sequence in the 3' untranslated region of epidermal growth factor messenger ribonucleic acid

Immunogenicity and protection studies with recombinant mycobacteria and vaccinia vectors coexpressing the 18-kilodalton protein of Mycobacterium leprae.

The activation of antigen-specific T lymphocytes is essential for the control of leprosy infection in humans and experimental animals. T cells recognize a variety of protein antigens from Mycobacterium leprae, including the 18-kDa protein, which is limited in distribution among mycobacteria and which is absent from Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG. Adjuvant preparations of mycobacterial protein antigens have had limited protective efficacy for experimental infections in animals. Since recombinant vectors may elicit more effective T-cell responses than adjuvant preparations, recombinant vaccinia virus (VV18) and M. bovis BCG (BCG18) vectors expressing the 18-kDa protein of M. leprae were prepared. Both VV18 and BCG18 stimulated anti-18-kDa protein antibody and lymphocyte proliferative responses. Sequential immunization with VV18 followed by BCG18 induced higher levels of specific immunoglobulin G2a antibodies than immunoglobulin G1 antibodies, in contrast to immunization with VV18 or BCG18 alone. The protective efficacy of immunization with VV18 from a challenge with BCG18 was examined in two murine models of mycobacterial infection. After intravenous challenge, mice immunized with recombinant vaccinia virus exhibited lower initial levels of replication and earlier clearance of BCG18 from their spleens than mice immunized with vaccinia virus expressing an unrelated protein. After footpad infection in a dissemination model, there was earlier clearance of BCG18 from specifically immunized mice. However, immunization of mice with VV18 did not prevent a productive mycobacterial infection.

Antibodies to a conserved-motif peptide sequence of the Plasmodium falciparum thrombospondin-related anonymous protein and circumsporozoite protein recognize a 78-kilodalton protein in the asexual blood stages of the parasite and inhibit merozoite invasion in vitro.

Athrombospondin-related anonymous protein (TRAP) of the human malaria parasite Plasmodium falciparum shares highly conserved amino acid sequence motifs with the circumsporozoite protein of all plasmodia sequenced so far, as well as with unrelated proteins like thrombospondin and properdin. Although it was first described as an asexual blood stages protein, there has been some controversy about its expression in these stages. Pursuant to our interest in the conserved sequences within the malaria antigens, we synthesized an 18-residue peptide (18-mer) representing a conserved motif of TRAP and raised polyclonal antibodies against it. In an immunoblot assay in which we probed proteins from the asexual blood stages of the parasite, we found that this antibody recognized predominantly a 78-kDa protein in the whole parasite lysate. Furthermore, in another immunoblot, the recombinant TRAP constructs containing the conserved-motif sequence were distinctly recognized by the antipeptide antibodies, whereas a construct lacking the motif sequence was not, suggesting that the antibodies specifically cross-reacted with a protein which might be a TRAP-like protein present in the asexual blood stages of the parasite. Also, in an immunofluorescence assay, this antibody brightly stained the acetone-fixed trophozoites of the parasite. Most significantly, anti-18-mer immunoglobulin G, as well as antipeptide antibody against a smaller (nonamer) construct representing the most conserved motif within the 18-mer, inhibited the merozoite invasion of erythrocytes in a dose-dependent manner. These results provide evidence of the expression of TRAP or a TRAP-like protein in the asexual blood stages of the parasite and of a possible role of the conserved motifs in the parasite-host cell interaction during the process of invasion.