Merozoite surface protein 8 of Plasmodium falciparum contains two epidermal growth factor-like domains.

By motif searching of the unfinished sequences in the Malaria Genome Sequencing Project databases we have identified a novel EGF-like domain-containing protein of Plasmodium falciparum. The sequence lies within a single open reading frame of 1791 bp and is predicted to encode a polypeptide of 597 amino acids. There are hydrophobic regions at the extreme N- and C-termini, which could represent secretory signal peptide and GPI attachment sites, respectively. Similar to MSP1, there are two EGF-like domains located near the C-terminus. RT-PCR analysis of the novel gene shows that it is transcribed in asexual stages of the malaria parasite. We have expressed portions of the protein as recombinant GST fusions in Escherichia coli and raised antisera in rabbits. Antibodies to the EGF-like domains of the novel protein are highly specific and do not cross-react with the EGF-like domains of MSP1, MSP4 or MSP5 expressed as GST fusion proteins. Antiserum raised to the most C-terminal region of the protein reacts with four bands of 98, 50, 25 and 19 kDa in P. falciparum parasite lysates whereas antisera to the N-terminal fusion proteins recognise the 98 and 50 kDa bands, suggesting that the novel protein may undergo processing in a similar way to MSP1. Immunoblot analysis of stage-specific parasite samples reveals that the protein is present throughout the parasite asexual life cycle and in isolated merozoites, with the smaller fragments present in ring stage parasites. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites, schizonts and free merozoites by indirect immunofluorescence. Antisera to the C-terminus stain the surface of rings, whereas antisera to the N-terminus do not, suggesting that a fragment of the protein is carried into the developing ring stage parasite. Based on the accepted nomenclature in the field we designate this protein MSP8. We have shown that the MSP8 fusion proteins are in a conformation that can be recognised by human immune sera and that there is very limited diversity in the MSP8 gene sequences from various P. falciparum laboratory isolates. MSP8 shows significant similarity to the recently reported sequence of the protective P. yoelii merozoite surface protein pypAg-2 [Burns JM, Belk CC, Dunn PD. Infect Immun 2000;68:6189-95.] suggesting that the two proteins are homologues. Taken together, these findings suggest that MSP8/pypAg-2 may play an important role in the process of red cell invasion and is a potential malaria vaccine candidate.

Lack of sequence diversity in the gene encoding merozoite surface protein 5 of Plasmodium falciparum.

The gene encoding merozoite surface protein 5 (MSP5) of Plasmodium falciparum is situated between the genes encoding MSP2 and MSP4 on chromosome 2. Both MSP4 and MSP5 encode proteins that contain hydrophobic signal and glycosylphosphatidylinositol (GPI) attachment signals and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. The similar gene organization, location and similar structural features of the two genes suggest that they have arisen from a gene duplication event. In this study we provide further evidence for the merozoite surface location of MSP5 by demonstrating that MSP5 is present in isolated merozoites, partitions in the detergent-enriched phase following Triton X-114 fractionation and shows a staining pattern consistent with merozoite surface location by indirect immunofluorescence confocal microscopy. Analysis of antigenic diversity of MSP5 shows a lack of sequence variation between various isolates of P. falciparum from different geographical locations, a feature unusual for surface proteins of merozoites and one that may simplify vaccine formulation.

A homologue of Sar1p localises to a novel trafficking pathway in malaria-infected erythrocytes.

We have identified a homologue of the GTP-binding protein, Sar1p, in Plasmodium falciparum. Sar1p is a small GTPase that is thought to play a crucial role in trafficking of proteins between the endoplasmic reticulum and the Golgi. The P.falciparum SAR1 gene is located on chromosome 4 and comprises two exons separated by a 508 bp intron. The deduced amino acid sequence of PfSar1p (GenBank accession number AF104306) shows 71% similarity (58% identity) to Sar1p from Saccharomyces cerevisiae. Expression of PfSar1p in erythrocytic stages of P. falciparum was confirmed by sequencing of a tryptic peptide derived from a polypeptide excised from an SDS-polyacrylamide gel. A recombinant protein corresponding to approximately 70% of the PfSar1p sequence was used to raise antibodies. The affinity-purified antiserum recognised a protein with an apparent molecular weight of 23 K in Western blots of malaria-infected erythrocytes but not in uninfected erythrocytes. PfSar1p was shown to be largely insoluble in non-ionic detergent and a low ionic strength buffer. Confocal immunofluorescence microscopy of malaria-infected erythrocytes was used to show that PfSar1p is located near the periphery of the parasite in discrete compartments, which appear to be distinct from the parasite endoplasmic reticulum. In addition, PfSar1p appears to be exported to structures outside the parasite in the erythrocyte cytoplasm. The export of PfSar1p to the erythrocyte cytosol is inhibited by treatment with brefeldin A. This provides the first evidence that the malaria parasite is capable of elaborating components of the classical vesicle-mediated trafficking machinery outside the boundaries of its own plasma membrane.

Identification of the Plasmodium chabaudi homologue of merozoite surface proteins 4 and 5 of Plasmodium falciparum.

Previous studies of Plasmodium falciparum have identified a region of chromosome 2 in which are clustered three genes for glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins, MSP2, MSP5, and MSP4, arranged in tandem. MSP4 and MSP5 both encode proteins 272 residues long that contain hydrophobic signal sequences, GPI attachment signals, and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. The locations and similar structural features of these genes suggest that they have arisen from a gene duplication event. Here we describe the identification of the syntenic region of the genome in the murine malaria parasite, Plasmodium chabaudi adami DS. Only one open reading frame is present in this region, and it encodes a protein with structural features reminiscent of both MSP4 and MSP5, including a single EGF-like domain. Accordingly, the gene has been designated PcMSP4/5. The homologue of the P. falciparum MSP2 gene could not be found in P. chabaudi; however, the amino terminus of the PcMSP4/5 protein shows similarity to that of MSP2. The PcMSP4/5 gene encodes a protein with an apparent molecular mass of 36 kDa, and this protein is detected in mature stages of the parasite. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites and developing and free merozoites. The PcMSP4/5 gene is transcribed in both ring and trophozoite stages but appears to be spliced in a stage-specific manner such that the central intron is spliced from the mRNA in the parasitic stage in which the protein is expressed.

Intranuclear localization of insulin-like growth factor binding protein-3 (IGFBP-3) during cell division in human keratinocytes.

Insulin-like growth factor-I (IGF-I) stimulation of basal keratinocytes is an essential component of normal epidermal homeostasis. In addition to the IGF receptor, basal keratinocytes synthesize insulin-like growth factor binding protein-3 (IGFBP-3). The HaCaT keratinocyte cell line, which has many characteristics of basal keratinocytes, synthesizes IGFBP-3 that in vitro reduces its IGF-I responsiveness. IGFBP-3 has attracted interest as a potential growth arrest protein, both via its ability to modulate IGF-I responsiveness, and more controversially via IGF-I-independent mechanisms. Intracellular modes of action have been proposed, and a nuclear localization consensus sequence has previously been identified within IGFBP-3. Using immunocytochemistry with a biotinylated antibody specific for IGFBP-3, we investigated the intracellular localization of IGFBP-3 in subconfluent monolayer cultures of HaCaT cells. Diffuse cellular staining was visible, potentially corresponding to cell surface and nascent cytoplasmic IGFBP-3. Of particular interest however, was the localization of staining over the nuclei of a large proportion of cells that were undergoing cell division. Antibody staining was specific for IGFBP-3 because addition of recombinant human IGFBP-3 to the antibody prior to incubation with the cells inhibited these staining patterns. Optical sections obtained using a confocal laser scanning microscope showed that in keratinocytes undergoing cell division, IGFBP-3 was localized inside the nucleus. These results show that intracellular IGFBP-3 localization is altered during the cell cycle and suggest a possible nuclear role for IGFBP-3 during cell division.

Identification of an endoplasmic reticulum-resident calcium-binding protein with multiple EF-hand motifs in asexual stages of Plasmodium falciparum.

An endoplasmic reticulum-located, calcium-binding protein, with an apparent molecular weight (Mr) of approximately 40,000 (PfERC), has been identified in the asexual stages of the malaria parasite, Plasmodium falciparum. This protein appears to be equivalent to a previously described gametocyte protein, Pfs40, which was reported to be expressed on the gametocyte surface (Rawlings DJ, Kaslow DC. J Biol Chem 1992;267:3976-3982). Sequencing of the 3' end of the gene revealed the omission of a single base in the 3' region of the published sequence. The corrected gene sequence encodes a C-terminal IDEL motif, which indicates residency of the 40 kDa protein within the endoplasmic reticulum. The predicted C-terminal region also appears to contain a sixth EF-hand calcium-binding domain, which suggests that PfERC is related to previously reported ER-localized calcium-binding proteins, namely reticulocalbin and ERC-55 (Ozawa M. J. Biochem. 1995;117:1113-1119; Weis K, Griffiths G, Lamond AI. J. Biol. Chem. 1994;269:19142-19150). The presence of the 40 kDa calcium-binding protein in malaria parasites was confirmed using 45Ca2+-blotting and partial protein sequencing of the corresponding Coomassie blue-stained polypeptide. Confocal immunofluorescence microscopy of asexual stage parasites was used to show that PfERC co-localizes with the known ER-located protein, Pfgrp. Analysis of immunoblots of tightly synchronized parasites showed that expression of PfERC increases with increasing maturity of the parasite. We propose that PfERC is a member of the reticulocalbin family of calcium-binding proteins and may play a role in protein trafficking in the malaria parasite.

A putative Plasmodium falciparum exported serine/threonine protein kinase.

An 8kb gene coding for a putative serine/threonine protein kinase from Plasmodium falciparum has been cloned and sequenced. It is arranged in two exons: exon I is 2 kb and exon II is 5.6 kb. The gene codes for a large protein of 2510 amino acids. Antibodies raised against a fusion protein were used to localize the putative kinase. By immunofluorescence microscopy, it was found in the cytoplasm of infected red cells. By immunoelectron microscopy it was associated with membranous structures in the red cell and with the red cell membrane, particularly at parasite-induced knobs. This is the first putative protein kinase of P. falciparum to be exported from the parasite into its host cell.

Macromolecular transport in malaria--does the duct exist?

The proposal that a parasitophorous 'duct' traverses the malaria-infected erythrocyte cytoplasm and is responsible for the unusual molecular uptake kinetics observed in malaria, has created considerable debate on the nature of macromolecular transport in this parasite. The existence of a 'duct' has important implications for the immunobiology of this parasite, particularly the possibility that antibodies may have access to 'internal' antigens in malaria. The most compelling evidence that there is a direct connection between the parasite and the surrounding media comes from the experiment of Pouvelle et al. (Nature 353, 73-75 (1991)) using small highly fluorescent latex spheres. However, we have found that fluorescent labeling of the parasite and tubular structures that extend from the parasite is due to the release of dye from the latex spheres during the incubation and is not due to the uptake of the spheres themselves. The inability of malaria-infected erythrocytes to take up latex beads down to 14 nm diameter establishes that an 'open' channel connecting the parasite with the surrounding media does not exist. This finding has important implications for establishing the unusual nature of macromolecular transport across the infected erythrocyte cytoplasm in malaria.

Babesia bovis: biosynthesis and localisation of 12D3 antigen in bovine erythrocytes.

The 12D3 antigen of Babesia bovis was found to be synthesised rapidly in cultured parasites, and localised to both the apical complex of the merozoite and the cytoplasm of the parasitised erythrocyte. Amino-terminal sequencing suggested that the nascent protein had been processed and differences between the predicted and measured molecular weights suggested post-translational modification. The major proportion of 12D3 appeared in the soluble compartment of the parasitised erythrocytes with a molecular weight consistent with no further processing. A significant proportion of the protein required extraction by sodium carbonate, suggesting association with membranous components. The timing of release of soluble 12D3 was coincident with haemoglobin release and this probably reflects a non-specific lysis of the erythrocyte. Synthesis of recombinant BV12D3 was achieved in baculovirus-infected SF9 insect epithelial cells. The product was of the same molecular weight as the native 12D3 and polyclonal antibodies raised against the recombinant protein reacted with both the recombinant and native forms of the antigen.

The endocytic pathway for H-ferritin established in live MOLT-4 cells by laser scanning confocal microscopy.

We have established the intracellular destination of the putative immunoregulatory protein, human recombinant H (heavy)-ferritin, in the transformed T-cell line MOLT-4, by laser scanning confocal microscopy of live cells. A series of confocal images was collected over a 60 min time course using indirect immunofluorescence of H-ferritin and transferrin, their respective monoclonal antibodies, and fluorescein isothiocyanate (FITC)-labelled IgG. A marked drop in FITC fluorescence after 40 min of H-ferritin internalization, indicative of an acidic environment, and co-localization with tetramethylrhodamine isothiocyanate-labelled-dextran strongly suggests that H-ferritin is transferred to the lysosome. In contrast, transferrin was observed to return to the cell surface. Electron microscopy confirmed that H-ferritin was transferred to the lysosome. The receptor-mediated endocytosis and lysosomal delivery of H-ferritin may thus potentiate its putative immunoregulatory activity.

Plasmodium falciparum: highly mobile small vesicles in the malaria-infected red blood cell cytoplasm.

A dynamic population of small vesicles within the cytoplasm of live malaria-infected red blood cells has been studied using a laser scanning confocal microscope. Acridine orange was used to follow the movement of vesicles within the infected red blood cell cytoplasm, including the budding of vesicles from the malarial parasite. These highly mobile vesicles are found predominantly in mid- to late ring-stage parasites and are almost entirely absent from young rings and mature trophozoites. Since the known parasite modifications of the red blood cell plasma membrane in mid-ring-to early trophozoite-stage parasites correlates with the appearance of acridine orange-staining vesicles, these vesicles may be an important component in the transport of parasite proteins across the infected red blood cell cytoplasm.

Subtype-specificity of antipeptide antibodies raised against unique sequences of human interferons-alpha.

A strategy for the production of human interferon-alpha (IFN-alpha) subtype-specific antibodies, based on immunizing rabbits with short unique synthetic peptides coupled to protein carriers, has been validated. These peptides correspond to amino acid residues 99-111 of IFN-alpha 1, 50-57 and 103-116 of IFN-alpha 2, and 37-50 of IFN-alpha 4. The antipeptide antibodies [anti-IFN alpha 1(99-111), anti-IFN alpha 2(50-57C), anti-IFN alpha 2(103-116) and anti-IFN alpha 4(C37-50)] were tested by ELISA and Western blotting for their reactivity with immunoaffinity-purified recombinant human IFN-alpha 1, -alpha 2b and -alpha 4a. The anti-IFN alpha 1(99-111) and anti-IFN alpha 2(50-57C) reacted with their corresponding IFN-alpha and did not crossreact with the other IFN subtypes. The anti-IFN alpha 2(103-116) reacted with IFN-alpha 2b and also crossreacted slightly with the other subtypes. The anti-IFN alpha 4(C37-50) reacted well with IFN-alpha 4a, crossreacted with significantly lower affinity with IFN-alpha 1 and did not bind IFN-alpha 2b. Residues 104-107 and 108-111 are the major components of the epitopes recognized by anti-IFN alpha 1(99-111) and anti-IFN alpha 2(103-116), respectively, as determined by ELISA against overlapping octapeptides.

Antipeptide antibodies against conserved regions of human interferon-alpha: evidence for conformational variations between IFN-alpha subtypes.

Antibodies to two conserved regions (residues 29-36 and 139-151) of human interferon-alpha were raised by immunizing rabbits with four short synthetic peptides coupled to carriers. The antibodies were tested for reactivity with recombinant interferon-alpha by ELISA. Despite the amino acid conservation of the two regions, there are significant variations in the reactivity of the antibodies with the IFN-alpha subtypes. The reactivity is enhanced significantly when the disulfide bonds of the interferon molecule are reduced. The results indicate that there are subtype-specific differences in the presentation of the epitopes in these conserved regions of human interferon-alpha.

A mathematical model of physiological processes and its application to the study of aging.

The behavior of a physiological system which, after displacement, returns by homeostatic mechanisms to its original condition can be described by a simple differential equation in which the "recovery time" is a parameter. Two such systems, which influence one another, can be linked mathematically by the use of "coupling" or "feedback" coefficients. These concepts are the basis for many mathematical models of physiological behavior, and we describe the general nature of such models. Next, we introduce the concept of a "fatal limit" for the displacement of a physiological system, and show how measures of such limits can be included in mathematical models. We show how the numerical values of such limits depend on the values of other system parameters, i.e., recovery times and coupling coefficients, and suggest ways of measuring all these parameters experimentally, for example by monitoring changes induced by X-irradiation. Next, we discuss age-related changes in these parameters, and show how the parameters of mortality statistics, such as the famous Gompertz parameters, can be derived from experimentally measurable changes. Concepts of onset-of-aging, critical or fatal limits, equilibrium value (homeostasis), recovery times and coupling constants are involved. Illustrations are given using published data from mouse and rat populations. We believe that this method of deriving survival patterns from model that is experimentally testable is unique.

In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation.

Mutants defective in the ability to translocate proteins across the membrane of the endoplasmic reticulum were selected in Trp- Saccharomyces cerevisiae on the basis of their ability to retain a fusion protein in the cytosol. The fusion comprised the prepro region of prepro-alpha-factor (MF alpha 1) N-terminal to phosphoribosyl anthranilate isomerase (TRP1). The first of the protein translocation mutations, called ptl1, results in temperature-sensitivity of growth and protein translocation. At the non-permissive temperature, precursors to several secretory proteins accumulate in the cytosol. Using this mutant, we demonstrate that the prepro-carboxypeptidase Y that had been accumulated in the cytosol at the non-permissive temperature could be post-translationally translocated into the endoplasmic reticulum when cells were returned to the permissive temperature. This result indicates that post-translational translocation of preproteins across endoplasmic reticulum membranes can occur in vivo. We have also determined that the temperature-sensitive component is membrane-associated in ptl1, and that the membranes derived from this strain show a reversible temperature-sensitive translocation phenotype in vitro.

Secretion in yeast: in vitro analysis of the sec53 mutant.

Of central importance to studying protein translocation via a combined genetic and biochemical approach is the in vitro analysis of yeast conditionally-lethal secretory mutants. Analysis of sec53 presented an opportunity not only to see if mutants could be examined in recently developed yeast in vitro translocation systems, but also to characterize further the nature of this mutant originally postulated to be defective in protein translocation. Membranes from sec53 were capable of translocating and glycosylating nascent prepro-alpha-factor in vitro in both sec53 and wild-type lysates at temperatures that were non-permissive for growth of the mutant cells. These results suggested that the Sec53 protein does not function directly in the translocation and glycosylation of prepro-alpha-factor. To examine this point further, we isolated membranes from sec53 cells that had been grown at the non-permissive temperature prior to disruption. In such cases, regardless of assay temperature, membranes from sec53 cells efficiently translocated but failed to glycosylate prepro-alpha-factor in vitro. The in vitro phenotype of sec53 could be mimicked by isolating rough microsomes from wild-type cells that had been grown for 1 h in the presence of tunicamycin. Together, these results demonstrate that sec53 is not defective in translocation, rather in assembly of the dolichol-oligosaccharide substrate needed for N-linked glycosylation.

Production of subtype-specific antipeptide antibodies to human interferon-alpha 1 and -alpha 4.

Antibodies that are specific to the human interferon (IFN)-alpha 1 and -alpha 4 subtypes have been produced by immunizing rabbits with two short synthetic peptides, corresponding to residues 99-111 of IFN-alpha 1 and residues 37-50 of IFN-alpha 4, respectively. The IFN-alpha 1 peptide has at least three closely clustered residues that are different from those in the other IFN-alpha subtypes, while the IFN-alpha 4 peptide has only two unique amino acid residues, separated by five common residues. The antibodies raised against the IFN-alpha 1 peptide react with recombinant human IFN-alpha 1 but do not cross-react with recombinant human IFN-alpha 4 or IFN-alpha 2. The antibodies raised against the IFN-alpha 4 peptide react with IFN-alpha 4, cross-react with IFN-alpha 1 but not with IFN-alpha 2; the affinity of the antibodies to IFN-alpha 1, however, is at least 10 times lower than their affinity to IFN-alpha 4.

Assembly of the mitochondrial ribosomes in a temperature-conditional mutant of Saccharomyces cerevisiae defective in the synthesis of the var1 protein.

An investigation of the role of the var1 protein in the assembly of the yeast mitochondrial ribosomes was carried out in a temperature conditional mutant, strain h56, which contains a mutation (tsv1) just upstream of the structural gene for the var1 protein. The mutation results in a marked decrease in the synthesis of the var1 protein at the permissive temperature of 28 degrees C and an apparently complete absence of var1 synthesis at the restrictive temperature of 36 degrees C. Long-term growth of strain h56 at the non-permissive temperature was found to result in the loss of the small (37 S) ribosomal subunit and the appearance of a novel 30 S ribonucleoparticle. Both the small (37 S) and the large (54 S) mitochondrial ribosomal subunits were found to be assembled in strain h56 for at least 3 h after transfer to the non-permissive temperature.

An assessment of the ability of yeast cells to incorporate photolabile fatty acids into their membrane phospholipids in vivo.

The photolabile fatty acids 12-azidooleic, 12-(4-azido-2-nitrophenoxy)oleic, 12-azidolauric and 12-(4-azido-2-nitrophenoxy)lauric are readily taken up in vivo by an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. A low level of the two lauric acid derivatives and none of the two oleic acid derivatives are incorporated into membrane phospholipids. Under certain conditions of growth in the presence of 12-(4-azido-2-nitrophenoxy)oleic acid, the nitrophenylazide group is metabolized to a product that lacks the photolabile azido group.

Are all mitochondrial translation products synthesized on membrane-bound ribosomes?

The Arrhenius kinetics for the synthesis of var1, which is a hydrophilic protein of the mitochondrial ribosomes, have been compared to that of other mitochondrial translation products, which are hydrophobic subunits of the respiratory enzyme complexes. Our results indicate that, in the yeast Saccharomyces cerevisiae, the hydrophilic var1 protein is synthesized on membrane-associated mitochondrial ribosomes which cannot be distinguished from those responsible for the synthesis of the hydrophobic mitochondrial translation products.