Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.     

The sperm outer dense fiber protein is the 10th member of the superfamily of mammalian small stress proteins

Nine proteins have been assigned to date to the superfamily of mammalian small heat shock proteins (sHsps): Hsp27 (HspB1, Hsp25), myotonic dystrophy protein kinase-binding protein (MKBP) (HspB2), HspB3, alphaA-crystallin (HspB4), alphaB-crystallin (HspB5), Hsp20 (p20, HspB6), cardiovascular heat shock protein (cvHsp [HspB7]), Hsp22 (HspB8), and HspB9. The most pronounced structural feature of sHsps is the alpha-crystallin domain, a conserved stretch of approximately 80 amino acid residues in the C-terminal half of the molecule. Using the alpha-crystallin domain of human Hsp27 as query in a BLAST search, we found sequence similarity with another mammalian protein, the sperm outer dense fiber protein (ODFP). ODFP occurs exclusively in the axoneme of sperm cells. Multiple alignment of human ODFP with the other human sHsps reveals that the primary structure of ODFP fits into the sequence pattern that is typical for this protein superfamily: alpha-crystallin domain (conserved), N-terminal domain (less conserved), central region (variable), and C-terminal tails (variable). In a phylogenetic analysis of 167 proteins of the sHsp superfamily, using Bayesian inference, mammalian ODFPs form a clade and are nested within previously identified sHsps, some of which have been implicated in cytoskeletal functions. Both the multiple alignment and the phylogeny suggest that ODFP is the 10th member of the superfamily of mammalian sHsps, and we propose to name it HspB10 in analogy with the other sHsps. The C-terminal tail of HspB10 has a remarkable low-complexity structure consisting of 10 repeats of the motif C-X-P. A BLAST search using the C-terminal tail as query revealed similarity with sequence elements in a number of Drosophila male sperm proteins, and mammalian type I keratins and cornifin-alpha. Taken together, the following findings suggest a specialized role of HspB10 in cytoskeleton: (1) the exclusive location in sperm cell tails, (2) the phylogenetic relationship with sHsps implicated in cytoskeletal functions, and (3) the partial similarity with cytoskeletal proteins.     

A heat shock protein localized to chloroplasts is a member of a eukaryotic superfamily of heat shock proteins.

We have isolated cDNA clones from soybean and pea that specify nuclear-encoded heat shock proteins (HSPs) which localize to chloroplasts. The mRNAs for these HSPs are undetectable at control temperatures, but increase approximately 150-fold during a 2-h heat shock. Hybridization-selection followed by in vitro translation demonstrates that these HSPs are synthesized as precursor proteins which are processed by the removal of 5-6.5 kd during import into isolated chloroplasts. The nucleotide sequence of the cDNAs shows the derived amino acid sequences of the mature pea and soybean proteins are 79% identical. While the predicted transit peptide encoded by the pea cDNA has some characteristics typical of transit sequences, including high Ser content, multiple basic residues and no acidic residues, it lacks two domains proposed to be important for import and maturation of other chloroplast proteins. The carboxy-terminal region of the chloroplast HSP has significant homology to cytoplasmic HSPs from soybean and other eukaryotes. We hypothesize that the chloroplast HSP shares a common structural and functional domain with low mol. wt HSPs which localize to other parts of the cell, and may have evolved from a nuclear gene.     

Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm

Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.     

Design and synthesis of a self-assembling peptide derived from the envelope proteins of HIV type 1. An approach to heterovalent immunogens.

A chimeric peptide that included sequences from gp120 and gp41 of HIV type 1 was synthesized. Cleavage from solid support yielded a composite of self-oligomerized products with molecular masses ranging from 5 to about 9 kDa. The oligomer but not its reduced, monomeric form was recognized by human anti-HIV sera and at least one of the two lysines in the sequence was involved in antibody binding. The oligomeric peptide was immunogenic, yielding a conformation-specific antibody response. Co-oligomerization of a hepatitis B surface Ag-derived peptide and the HIV type 1-derived peptide yielded a bivalent product in which conformational integrity of the individual components was maintained. Immunization with this hybrid peptide resulted in conformation-specific antibodies to both epitopes in all four murine strains tested. Lymphocyte proliferation assays revealed that the T epitopes resident in both peptide sequences remained active in the hybrid peptide. These results demonstrate the potential of this approach in generating multi- and heterovalent immunogens which may eventually find application as vaccines.     

Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family

We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically. © 1996 Wiley-Liss, Inc.     

MAGE-F1, a novel ubiquitously expressed member of the MAGE superfamily

Most known members of the MAGE superfamily are expressed in tumors, testis and fetal tissues, which has been described as a cancer/testis or "CT" expression pattern. We have identified a novel member of this superfamily, MAGE-F1, which is expressed in all adult and fetal tissues tested. In addition to normal tissues, MAGE-F1 is expressed in many tumor types including ovarian, breast, cervical, melanoma and leukemia. MAGE-F1 is encoded on chromosome 3, identifying a sixth chromosomal location for a MAGE superfamily gene. The coding region of MAGE-F1 is contained within a single exon and includes a microsatellite repeat. Sequence analysis and expression profiles define a new class of ubiquitously expressed MAGE superfamily genes that includes MAGE-F1, MAGE-D1, MAGE-D2/JCL-1 and NDN. The finding that several MAGE genes are ubiquitously expressed suggests a role for MAGE encoded proteins in normal cell physiology. Furthermore, potential cross-reactivity to these ubiquitously expressed MAGE gene products should be considered in the design of MAGE-targeted immunotherapies for cancer.     

Calmodulin-binding protein BP-10, a probable new member of plant nonspecific lipid transfer protein superfamily.

CaMBP-10 is a novel plant endogenous calmodulin-binding protein with important physiological functions. The partial cDNA sequence of this protein was cloned using RT-PCR. The deduced peptide (designated PCBP10) is composed of 74 amino acid residues containing a basic amphiphilic alpha-helix typical for calmodulin-binding proteins. PCBP10 shows very high amino acid sequence homology with plant nonspecific lipid-transfer proteins (nsLTPs). Sequence analysis also reveals that PCBP10 has similar amino acid composition to plant nsLTPs, and seven of the eight conserved cysteine residues are found in PCBP10. Furthermore, the secondary structure features of PCBP10 are very similar to those of plant nsLTPs. In addition, there are striking resemblances between CaMBP-10 and plant nsLTPs in their biochemical and physical properties. Our results suggest that CaMBP-10 is a novel member of the plant and nsLTP gene family, and the Ca(2+)/CaM regulative system may also play roles in lipid metabolism, defense reactions, and the adaptation of plants to natural environment.     

BH-protocadherin-c, a member of the cadherin superfamily, interacts with protein phosphatase 1 alpha through its intracellular domain

Using a yeast two-hybrid system, we isolated eight cDNA clones which interacted with BH-protocadherin-c (BH-Pcdh-c) from the human brain cDNA library. One clone encoded protein phosphatase type I isoform alpha (PP1alpha) and another two PP1alpha2. PP1alpha was co-immunoprecipitated from the extract of a gastric adenocarcinoma cell line MKN-28 with anti-BH-Pcdh-c antibody. PP1alpha activity towards glycogen phosphorylase was inhibited by the intracellular domain of BH-Pcdh-c. Inhibition of the phosphatase required more than the minimal domain of BH-Pcdh-c which could associate with PP1alpha. In situ hybridization revealed that BH-Pcdh-c mRNA was predominantly expressed in cerebral cortex neurons in the adult mouse brain.     

PFA, a novel mollusk agglutinin, is structurally related to the ribosome-inactivating protein superfamily

The structural organization of PFA, a novel beta-galactose-specific agglutinin from the snail Pomacea flagellata, was partially characterized. Using mass spectrometry, the molecular weight of this glycoprotein was determined as 32,444 Da (7.4% carbohydrate). The agglutinin was found to form very large aggregates in solution, which were dissociated to monodisperse native-like dimers upon addition of polyethyleneglycol. The identity of the first 38 and the last 11 residues of the polypeptide chain was determined. It was found that PFA and the N-glycosidase subunit of ricin, a heterodimeric cytotoxin isolated from castor bean seeds, are homologous to each other in the N-terminal region. Furthermore, the far-UV circular dichroism spectra of these proteins were found to be nearly superimposable, evidencing that they share common general features in their secondary and tertiary structures. On the basis of these similarities, it can be concluded that PFA is structurally related to the ribosome-inactivating protein superfamily.     

Phg1p is a nine-transmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis

To identify the molecular mechanisms involved in phagocytosis, we generated random insertion mutants of Dictyostelium discoideum and selected two mutants defective for phagocytosis. Both represented insertions in the same gene, named PHG1. This gene encodes a polytopic membrane protein with an N-terminal lumenal domain and nine potential transmembrane segments. Homologous genes can be identified in many species; however, their function is yet to be elucidated. Disruption of PHG1 caused a selective defect in phagocytosis of latex beads and Escherichia coli, but not Klebsiella aerogenes bacteria. This defect in phagocytosis was caused by a decrease in the adhesion of mutant cells to phagocytosed particles. These results indicate that the Phg1 protein is involved in the adhesion of Dictyostelium to various substrates, a crucial event of phagocytosis and demonstrate the usefulness of a genetic approach to dissect the molecular events involved in the phagocytic process.     

cDNA cloning and sequencing reveals human tear prealbumin to be a member of the lipophilic-ligand carrier protein superfamily.

The gene encoding human tear prealbumin, a major component of the protein fraction of tear fluid, was cloned from total cDNA of lacrimal gland by polymerase chain reaction using synthetic oligonucleotides derived from N-terminal amino acid sequences of the purified protein. Sequence analysis and a computer-assisted homology search revealed this protein to be a member of the lipocalin superfamily, consisting of hydrophobic-ligand carriers. The deduced amino acid sequence of tear prealbumin shares 58% identity with von Ebner's gland protein from rat, which is supposed to be involved in taste reception. In addition, significant homology has also been found with other members of the lipocalins, e.g. with beta-lactoglobulin. The predicted secondary structure of tear prealbumin resembles that of beta-lactoglobulin in the number and positions of nine beta-sheets and one alpha-helix at the C-terminal part of the protein, thus indicating a three-dimensional structure similar to beta-lactoglobulin. Protein sequencing revealed that the observed electrophoretic heterogeneity of tear prealbumin is due to subtle differences at the N terminus of the protein. The function of tear prealbumin as a lipophilic carrier was further supported by the fact that it binds [3H]retinol in vitro. Although this protein was originally described to be tear-specific, a tear prealbumin-specific antiserum also reacted with proteins of human saliva, sweat, and nasal mucus.