Co- and post-translational processing of the hevein preproprotein of latex of the rubber tree (Hevea brasiliensis)

Hevein is a chitin-binding protein of 43 amino acids found in the lutoid body-enriched fraction of rubber tree latex. A hevein cDNA clone (HEV1) (Broekaert, W., Lee, H.-i., Kush, A., Nam, C.-H., and Raikhel, N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7633-7637) encodes a putative signal sequence of 17 amino acids followed by a polypeptide of 187 amino acids. Interestingly, this polypeptide has two distinct domains: an amino-terminal domain of 43 amino acids, corresponding to mature hevein, and a carboxyl-terminal domain of 144 amino acids. To investigate the mechanisms involved in processing of the protein encoded by HEV1, three domain-specific antisera were raised against fusion proteins harboring the amino-terminal domain (N domain), carboxyl-terminal domain (C domain), and both domains (NC domain). Translocation experiments using an in vitro translation system show that the first 17-amino acid sequence encoded by the cDNA functions as a signal peptide. Immunoblot analysis of proteins extracted from lutoid bodies demonstrates that a 5-kDa protein comigrated with purified mature hevein and cross-reacted with N domain- and NC domain-specific antibodies. A 14-kDa protein was recognized by C domain- and NC domain-specific antibodies. A 20-kDa protein was cross-reactive with all three antibodies. Microsequencing data further suggest that the 5-kDa (amino-terminal domain) and 14-kDa (carboxyl-terminal domain) proteins are post-translational cleavage products of the 20-kDa polypeptide (both domains) which corresponds to the proprotein encoded by HEV1. In addition, it was found that the amino-terminal domain could provide chitin-binding properties to a fusion protein bearing it either amino terminally or carboxyl terminally.     

Characterization of the subtilase gene family in tomato (Lycopersicon esculentum Mill.)

The gene family of subtilisin-like serine proteases (subtilases, SBTs) in tomato (Lycopersicon esculentum Mill.) comprises at least 15 members, 12 of which have been characterized in this study. Sequence comparison revealed that tomato subtilases fall into 5 distinct subfamilies. Single genes were shown to exist for LeSBT1, LeSBT2 and tmp, while 5 and 6 genes were found in the LeSBT3/4 and P69 subfamilies, respectively. With the exception of tmp, tomato subtilase genes were found to lack introns. Expression of subtilase genes was confirmed at the mRNA level by northern blot analysis and/or by primer extension experiments. For each of the 5 subtilase subfamilies, a distinctive pattern of expression was observed in tomato organs. At least one of the subtilases was found to be expressed in each organ analysed. Structural features evident from deduced amino acid sequences are discussed with reference to the related mammalian proprotein convertases.     

Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K

A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.     

Expression of human prohormone convertase PC2 in a baculovirus-insect cell system.

PC2 is a member of the eukaryotic family of subtilisin-related proprotein convertases which are thought to be involved in the intracellular proteolytic processing of prohormones and proneuropeptides. The presence of only small amounts of PC2 in the secretory granules of certain mammalian neuroendocrine cell types has made the characterization and further study of this enzyme difficult. We report here the expression of proteolytically active human PC2 protein in the insect cell-baculovirus system. Human PC2 expressed in insect cells is a calcium-dependent intracellular protein active at neutral pH. In insect cells, human PC2 was found intracellularly as 75-kDa and 71-kDa proteins. Both 73-kDa and 68-kDa forms were found in the conditioned medium, but no PC2 proteolytic activity was detected. We demonstrated the presence of a soluble inhibitor in infected-cell medium which may block PC2 activity.     

Cloning and expression of human 75-kDa inositol polyphosphate-5-phosphatase.

Inositol polyphosphate-5-phosphatase (5-phosphatase) hydrolyzes inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thereby functions as a signal terminating enzyme in cellular calcium ion mobilization. A cDNA encoding human platelet 5-phosphatase has been isolated by screening for beta-galactosidase fusion proteins that bind to inositol 1,3,4,5-tetrakisphosphate. The sensitivity of the screening procedure was enhanced 50- to 100-fold by amplification of "sublibraries" prior to carrying out binding assays. The sequences derived from the "expression clone" were used to screen human erythroleukemia cell line and human megakaryocytic cell line cDNA libraries. We obtained two additional clones which together consist of 2381 base pairs. The amino-terminal amino acid sequence from the 75-kDa 5-phosphatase purified from platelets is identical to amino acids 38-56 predicted from the cDNA. This suggests that the platelet 5-phosphatase is formed by proteolytic processing of a larger precursor. The cDNA predicts that the mature enzyme contains 635 amino acids (Mr 72, 891). Antibodies directed against recombinant TrpE fusion proteins of either an amino-terminal region or a carboxyl-terminal region immunoprecipitate the enzyme activity from a preparation of the 75-kDa form of platelet 5-phosphatase (Type II) but do not precipitate the distinct 47-kDa 5-phosphatase (Type I) also found in platelets. In addition, the recombinant protein expressed in Cos-7 cells has the same 5-phosphatase activity as the platelet 5-phosphatase.     

A multicomponent, elicitor-inducible cystatin complex in tomato, Solanum lycopersicum

We assessed the ability of the fungal elicitor arachidonic acid to induce cystatin genes in tomato (Solanum lycopersicum), using a cDNA expression library from arachidonate-treated leaves. The cDNAs of two novel cystatins were isolated, coding for an approx. 11-kDa protein, SlCYS10; and for a 23.6-kDa protein, SlCYS9, bearing an N-terminal signal peptide and a long, 11.5-kDa extension at the C terminus. Both genes were induced by arachidonate but not by methyl jasmonate, an inducer of the 88-kDa eight-unit cystatin, multicystatin, accumulated in the cytosol of leaf cells upon herbivory. A truncated form of SlCYS9, tSlCYS9, was produced by deletion of the C-terminal extension to assess the influence of this structural element on the cystatin moiety. As shown by kinetic and stability assays with recombinant variants expressed in Escherichia coli, deleting the extension influenced both the overall stability and inhibitory potency of SlCYS9 against cysteine proteases of herbivorous organisms. These findings provide evidence for a multicomponent elicitor-inducible cystatin complex in tomato, including at least 10 cystatin units produced via two metabolic routes.     

Processing of rabbit hemorrhagic disease virus polyprotein.

Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lysates and in Escherichia coli have been used to study the proteolytic processing of RHDV polyprotein encoded by ORF1. An epitope tag was used for monitoring the gene products by a specific antibody. We have identified four gene products with molecular masses of 80, 43, 73, and 60 kDa, from the amino to the carboxy terminus of the polyprotein. The amino-terminal sequences of the 43- and 73-kDa products were determined and indicated that RHDV 3C proteinase cleaved Glu-Gly peptide bonds.     

番茄COBRA基因克隆、表达模式及生物信息学分析

COBRA作为一种重要的胞外糖基磷脂酰肌醇(GP-I)锚定蛋白,影响植物细胞壁中纤维素含量及细胞的定向伸长。目前已有多个拟南芥、玉米及水稻的COBRA基因的突变体被研究。而有关番茄COBRA基因克隆的研究尚未见报道。本研究利用RT-PCR技术克隆了一个假定编码番茄COBRA蛋白的SlCOBRA基因,并在GenBank注册(JN398667)。序列测定和分析表明,该序列由6个外显子组成,编码444个氨基酸残基;氨基酸序列中存在COBRA蛋白的CCVS保守基序,N端的跨膜信号肽及C-末端的疏水性尾部和GPI锚定ω-位点。系统进化分析表明番茄SlCOBRA与拟南芥AtCOB具有80%氨基酸序列同源性,聚在一个分支上。Real-time PCR分析番茄各个组织中COBRA基因的表达结果表明番茄COBRA为组成型表达,在营养器官(根、茎、叶)中的表达量高于花和果实,尤其在成熟的果实中(从转色期到红熟期)表达量明显减少。     

cDNA-derived primary structure of the glycoprotein component of canine microsomal signal peptidase complex

Canine microsomal signal peptidase activity has been shown previously to co-migrate as an apparent complex of six polypeptides with molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa species are differentially glycosylated forms of the same protein, designated SPC 22/23. The amino acid sequence of SPC 22/23 was deduced from cDNA clones. The protein is synthesized without a cleavable amino-terminal signal sequence and contains a single site for N-linked glycosylation. SPC 22/23 appears to be anchored to the rough endoplasmic reticulum membrane by a single hydrophobic segment near its amino terminus, with the remainder of the protein positioned on the lumenal side of the membrane. The amino acid sequence of SPC 22/23 shares homology with tryptic peptides derived from the hen oviduct signal peptidase glycoprotein, one of two possible proteins required for signal peptide processing in the avian system (Baker, R.K., and Lively, M.O. (1987) Biochemistry 26, 8561-8567). Therefore, the complete amino acid sequence of SPC 22/23 presented in this report corresponds to one of two possible proteins required for signal peptide processing in higher eukaryotic cells.     

Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines.

The cDNA encoding human myeloperoxidase carries three ATG codons in frame; 144, 111 and 66 bp upstream from the proprotein DNA sequence. In order to determine the most efficient signal sequence, three cDNA modules starting at each of the ATG were cloned into an eucaryotic expression vector and stably expressed in Chinese hamster ovary cell lines. In all three cases, recombinant human myeloperoxidase (recMPO) was secreted into the culture medium of transfected cells, indicating that each of the signal peptides functions efficiently. One of the recombinant cell lines, which was amplified using methotrexate, overexpresses enzymatically active recMPO up to 6 micrograms.ml-1.day-1. The recombinant product was purified by a combination of ion-exchange and metal-chelate chromatography, and characterized in terms of molecular mass, amino-terminal amino acid analysis, glycosylation, physicochemical properties and biological activity. The data show that recMPO is secreted essentially as a monomeric, heme-containing, single-chain precursor of 84 kDa which exhibits peroxidase activity. Amino-terminal analysis indicated that cleavage of the signal peptide occurs between amino acids 48 and 49. In addition, recMPO appeared to be glycosylated up to the last stage of sialylation, to an extent similar to that of the natural enzyme. Specific activity measurements as well as stability data, in various pH, temperature, ionic strength and reducing conditions, indicated that the recombinant single-chain enzyme behaves essentially in the same way as the natural two-chain molecule. Finally, recMPO was shown to exert potent cytotoxicity towards Escherichia coli when provided with its physiological substrates, i.e. hydrogen peroxide and chloride ions.     

Characterization and expression of an antifungal zeamatin-like protein (Zlp) gene from Zea mays.

A cDNA clone encoding a basic thaumatin-like protein of Zea mays was recovered from a mid-development seed cDNA library. The gene, Zlp, encoded a protein that was nearly identical with maize zeamatin and alpha-amylase/trypsin inhibitor. Expression of Zlp mRNA was highest in the endosperm tissue of seed 4 weeks after pollination. Expression of zeamatin-like (ZLP) protein correlated with mRNA; also, a low basal level of ZLP expression in leaf was not appreciably induced by abiotic stresses. ZLP was expressed with its own signal peptide in insect cells and in transgenic Arabidopsis and tomato plants. ZLP was secreted in all three systems, with correct processing of the signal peptide. ZLP expressed in transgenic tomato was found to be partially subjected to a proteolytic cleavage after residue 180, by an unknown mechanism, to give a "nicked" isoform of ZLP. Purified ZLP from all three sources, as well as purified "nicked" ZLP from tomato, demonstrated fungal inhibition against Candida albicans and Trichoderma reesei, with marginal inhibition observed against Alternaria solani and Neurospora crassa.     

Processing of pulmonary surfactant protein B by napsin and cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.     

Expression of biotin-binding proteins,avidin and streptavidin,in plant tissues using plant vacuolar targeting sequences

Tobacco plants have been developed which constitutively express high levels of the biotin-binding proteins, avidin and streptavidin. These plants were phenotypically normal and produced fertile pollen and seeds. The transgene was expressed and its product located in the vacuoles of most cell types in the plants. Targeting was achieved by use of N-terminal vacuolar targeting sequences derived from potato proteinase inhibitors which are known to target constitutively to vacuoles in potato tubers and, under wound-induction, in tomato leaves. Avidin was located in protein body-like structures within the vacuole and transgene protein levels remained relatively constant throughout the lifetime of the leaf. We describe two chimeric constructs with similar levels of expression. One comprised a potato proteinase inhibitor I signal peptide cDNA sequence attached to an avidin cDNA and the second a potato proteinase inhibitor II signal peptide genomic sequence (including an intron) attached to a core streptavidin synthetic sequence. We were unable to regenerate plants when transformation used constructs lacking the targeting sequences. The highest levels observed (up to 1.5% of total leaf protein) confirm the vacuole as the organelle of choice for stable storage of plant-toxic transgene products. The efficient targeting of these proteins did not result in any measured changes in plant biotinmetabolism.     

cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues.

A cDNA encoding the human fur gene product was isolated from a human hepatoma cell line. The cDNA encodes a protein with significant amino acid sequence identity to the prokaryotic subtilisin family of serine proteases. More extensive sequence identity was found when the protein was compared with eukaryotic proteases such as PRB1 of Saccharomyces cerevisiae, and with PC2 and PC3, the only other known mammalian subtilisin-like proteases. In contrast to these proteins, however, the fur gene product shares a more extensive topographic and functional homology with the KEX2 endoprotease of S. cerevisiae. Each protease contains a signal peptide, a glycosylated extra cytoplasmic domain, a hydrophobic membrane-spanning region, and a short, hydrophilic "tail" sequence. As with KEX2, the expressed human protease was shown to cleave mammalian proproteins at their paired basic amino acid processing sites. We have, therefore, proposed the function-based acronym PACE (paired basic amino acid cleaving enzyme) for this prototypic mammalian proprotein processing enzyme.     

Characterization of pollen polygalacturonase encoded by several cDNA clones in maize

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes form a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.     

Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211bp, consisting of a 5'-terminal untranslated region (UTR) of 72bp, a 3'-terminal UTR of 77bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16h after injury and injection of bacteria.