Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme

Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino acid sequence of batroxobin for the first time for a thrombin-like snake venom enzyme. The molecular weight of batroxobin is 25,503 (231 amino acids). The amino acid sequence of batroxobin exhibits significant homology with those of mammalian serine proteases (trypsin, pancreatic kallikrein, and thrombin), indicating that batroxobin is a member of the serine protease family. Based on this homology and enzymatic and chemical studies, the catalytic residues and disulfide bridges of batroxobin were deduced to be as follows: catalytic residues, His41, Asp86, and Ser178; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163, and Cys174-Cys199. The amino-terminal amino acid residue of batroxobin, valine, is preceded by 24 amino acids. This may indicate that the amino-terminal hydrophobic peptide (18 amino acids) is a prepeptide and that the hydrophilic peptide (6 amino acids), preceded by the putative prepeptide, is a propeptide.     

Adaptive evolution in the snake venom Kunitz/BPTI protein family

Snake venoms are rich sources of serine proteinase inhibitors that are members of the Kunitz/BPTI (bovine pancreatic trypsin inhibitor) family. However, only a few of their gene sequences have been determined from snakes. We therefore cloned the cDNAs for the trypsin and chymotrypsin inhibitors from a Vipera ammodytes venom gland cDNA library. Phylogenetic analysis of these and other snake Kunitz/BPTI homologs shows the presence of three clusters, where sequences cluster by functional role. Analysis of the nucleotide sequences from the snake Kunitz/BPTI family shows that positive Darwinian selection was operating on the highly conserved BPTI fold, indicating that this family evolved by gene duplication and rapid diversification.     

A novel prothrombin activator from the venom of Micropechis ikaheka: isolation and characterization

A novel prothrombin activator, Mikarin, has been isolated from Micropechis ikaheka venom. It is a single polypeptide chain metalloproteinase with the apparent molecular weight of 47kDa. Mikarin exhibits Ca(2+)-independent prothrombin activation, but no effects on other blood coagulation factors, such as factor X and fibrinogen. Mikarin is the first member of group I prothrombin activators from elapid venom. Like other high-molecular-weight snake venom proteinases, it has three structural domains, metalloproteinase and disintegrin-like and Cys-rich domains, and belongs to the P-III class of snake venom metalloproteinases. The N-terminal of Mikarin exhibits 76% sequence identity with Cobrin, a metalloproteinase identified from Naja naja venom, but very lower identities were found when compared with those from viperid and crotalid venom. In addition, the presence of disintegrin-like and Cys-rich domains in snake venom metalloproteinases with diverse biological activities suggests that these domains may be important for their function.     

Immunological identification of rat tissue kallikrein cDNA and characterization of the kallikrein gene family

A tissue kallikrein cDNA was identified by direct immunological screening with affinity-purified anti-rat tissue kallikrein antibody from a rat submandibular cDNA library constructed with the expression vector pUC8. Sequence analysis of the kallikrein cDNA revealed an encoded protein 97% homologous to the partial amino acid sequence of rat submandibular kallikrein. This cDNA was used to hybrid-select kallikrein-specific RNA from submandibular gland. Translation of the hybrid-selected RNA in a cell-free assay system resulted in the production of a 37 kDa peptide representing the preproenzyme. In addition, hybrid-selection of RNA under less stringent conditions showed cross-hybridization with other submandibular gland mRNA species. In correlation with these results, analysis of rat genomic DNA showed extensive hybridization, suggesting a family of closely related kallikrein-like genes. Consequently, a Charon 4A rat genomic library was screened for kallikrein genes by hybridization with rat tissue kallikrein cDNA. Thirty-four clones were isolated and found to be highly homologous by hybridization and restriction enzymes analyses. Fourteen unique clones were identified by restriction enzyme site polymorphisms within DNA segments which hybridized to the kallikrein cDNA probe and it was estimated that at least 17 different kallikrein-like genes are present in the rat. Sequence and structural analysis of one of the genomic clones revealed a gene structure similar to that of other serine proteinases. Comparison of the partially sequenced exon regions of the gene with the sequence of rat tissue kallikrein cDNA reveals 89% identity when aligned for the greatest homology. However, the genomic sequence predicts termination codons in all three translational reading frames, implying that this gene is nonfunctional, i.e., a pseudogene. Comparison of the rat genomic sequence to a kallikrein-like gene from the mouse reveals extensive preservation of exons, less identity within introns and no significant homology between extragenic regions.     

The expression of two kallikrein gene family members in the rat kidney

The mRNAs for two kallikrein gene family members expressed in the rat kidney have been characterized. One mRNA (PS) has previously been found in the pancreas and submaxillary gland and encodes true kallikrein. The second mRNA (K1) encodes a novel kallikrein-like enzyme expressed in the kidney and submaxillary gland that retains many of the key amino acid residues for the characteristic enzymatic cleavage specificity of kallikrein. Two oligonucleotide hybridization probes specific for the K1 mRNA demonstrate that the K1 mRNA is expressed in the kidney and submaxillary gland, but in none of the other eight tissues known to express one or more members of the rat kallikrein gene family. The K1 mRNA is the dominant kallikrein-related mRNA of the kidney, expressed at roughly 10 times the level of the true kallikrein (PS) mRNA. In the submaxillary gland the K1 mRNA is expressed at roughly one-fourth the level of true kallikrein mRNA.     

Proteinase inhibitors and dendrotoxins. Sequence classification, structural prediction and structure/activity

The amino acid sequences of four presynaptically active toxins from mamba snake venom (termed 'dendrotoxins') were compared systematically with homologous sequences of members of the proteinase inhibitor family (Kunitz). A comparison based on the complete sequences revealed that relatively few amino acid changes are necessary to abolish antiprotease activity and convert a proteinase inhibitor into a dendrotoxin. When comparison centred only on the sequence segments known to comprise the antiprotease site of bovine pancreatic trypsin inhibitor, the dendrotoxins were clearly classified apart from all the known inhibitors. Since the mode of action of the bovine pancreatic trypsin/kallikrein inhibitor involves beta sheet formation with the enzyme, predictions were obtained for this secondary structure in the region of the 'antiprotease site' throughout the homologues. Again, the dendrotoxins were clearly distinguished from the inhibitors. Structure/activity analyses, based on the crystal structures of inhibitor/enzyme complexes, suggest that unlike proteinase inhibitors, dendrotoxins might specifically co-ordinate the active-site 'catalytic' histidine residues of serine proteases. Although the significance of this remains to be studied, the presynaptic target is expected to involve an as yet uncharacterised member of the serine protease family.     

Distinctly different gene structure of KLK4/KLK-L1/prostase/ARM1 compared with other members of the kallikrein family: intracellular localization, alternative cDNA forms, and Regulation by multiple hormones

The tissue kallikreins (KLKs) form a family of serine proteases that are involved in processing of polypeptide precursors and have important roles in a variety of physiologic and pathological processes. Common features of all tissue kallikrein genes identified to date in various species include a similar genomic organization of five exons, a conserved triad of amino acids for serine protease catalytic activity, and a signal peptide sequence encoded in the first exon. Here, we show that KLK4/KLK-L1/prostase/ARM1 (hereafter called KLK4) is the first significantly divergent member of the kallikrein family. The exon predicted to code for a signal peptide is absent in KLK4, which is likely to affect the function of the encoded protein. Green fluorescent protein (GFP)-tagged KLK4 has a distinct perinuclear localization, suggesting that its primary function is inside the cell, in contrast to the other tissue kallikreins characterized so far that have major extracellular functions. There are at least two differentially spliced, truncated variants of KLK4 that are either exclusively or predominantly localized to the nucleus when labeled with GFP. Furthermore, KLK4 expression is regulated by multiple hormones in prostate cancer cells and is deregulated in the androgen-independent phase of prostate cancer. These findings demonstrate that KLK4 is a unique member of the kallikrein family that may have a role in the progression of prostate cancer.     

Structure of the gene for human complement protein decay accelerating factor.

Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane protein that is part of the regulators of complement activation (RCA) gene family located on human chromosome 1, band q32. These proteins, beginning at their amino terminus, consist largely of multiple copies of an approximately 60 amino acid short consensus repeat (SCR). A DAF cDNA clone was used to identify overlapping bacteriophage genomic clones. The human DAF gene spans approximately 40 kb and consists of 11 exons. The length of these exons and introns varies considerably, with the exons ranging from 21 to 956 bp and the introns ranging from approximately 0.5 to 19.8 kb. SCR I, II, and IV are all encoded by single exons; however, SCR III is encoded by two separate exons, with the splice junction occurring after the second nucleotide of the codon for the glycine residue at position 34 of the consensus sequence. This feature has also been found in CR1, CR2, membrane cofactor protein, and murine factor H. Following the SCR in DAF is a 76 amino acid serine/threonine-rich domain encoded on three separate exons. Exon 10 encodes the Alu family sequence that has been found as an insert in a minor class of DAF cDNA, thus indicating that this mRNA arises by standard alternative splicing. The last DAF exon, which comes after the largest intron of 19.8 kb, encodes the hydrophobic carboxy terminus and the 3'UT region. The nature of the signal that directs posttranslational attachment of a glycophospholipid anchor to DAF is not known, but that signal is apparently spread over three exons and greater than 20 kb. An analysis of the DAF gene provides additional evidence for the common evolutionary heritage of the RCA gene family. The exon/intron structure of this gene will facilitate experiments aimed at understanding the functions of the various domains of DAF.     

Structurally conserved alpha-neurotoxin genes encode functionally diverse proteins in the venom of Naja sputatrix

The structure and organization of the genes encoding the long-chain neurotoxins and four other isoforms of weak neurotoxins in the venom of Naja sputatrix are reported. The genes contained three exons interrupted by two introns, a structure similar to other members of the three-finger toxin family. The proteins encoded by these genes, however, show varied affinity towards nicotinic acetylcholine receptors. Phylogenetic analysis of these genes showed that the weak neurotoxin gene is confined to a distinct group. We also observe that specific mutations of the gene provide the diversity in function in these toxins while maintaining a common structural scaffold. This forms the first report where the molecular basis of evolution of postsynaptic neurotoxins from an ancestral gene can be demonstrated using the same species of snake.     

长白山白眉蝮蛇乌苏里亚种类凝血酶（TLE）基因克隆及分析

目的为了获得长白山白眉蝮蛇乌苏里亚种类凝血酶基因。方法根据GeneBank自眉类凝血酶eDNA5．和3保守序列设计了引物,通过RT-PCR从白眉蝮蛇乌苏里亚种毒腺TotalRNA中扩增得到1条长714bp的特异cDNA片段,将该cDNA片段重组到SimpleTvector,转化进E．coliJM109competentcell,阳性克隆委托生物公司测序,利用生物信息学方法对测序结果进行分析。结果该特异性片段与蛇毒类凝血酶同源性为95％,它为一个开发阅读框架,其编码的蛋白质序列与其他蛇毒类凝血酶序列同源性为94％,与其他蛇毒类凝血亲缘关系非常近。结论本实验获得了一种新型白眉蝮蛇乌苏里亚种类凝血酶基因。     

KLK12 is a novel serine protease and a new member of the human kallikrein gene family-differential expression in breast cancer

Kallikreins are a subgroup of serine proteases that are involved in the posttranslational processing of polypeptide precursors. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins are encoded by a large multigene family, but in humans, only three genes have been identified. By using the positional candidate approach, we were able to identify a new kallikrein-like gene, tentatively named KLK12 (for kallikrein gene 12). This new gene maps to chromosome 19q13.3-q13.4, is formed of five coding exons, and shows structural similarity to serine proteases and other known kallikreins. KLK12 is expressed in a variety of tissues including salivary gland, stomach, uterus, lung, thymus, prostate, colon, brain, breast, thyroid, and trachea. We identified three splicing forms of KLK12 that are expressed in many tissues. Our preliminary results indicate that the expression of KLK12 is down-regulated at the mRNA level in breast cancer tissues and is up-regulated by steroid hormones in breast and prostate cancer cell lines. This gene may be involved in the pathogenesis and/or progression of certain cancer types and may find applicability as a novel cancer biomarker.     

Glandular kallikreins of the cotton-top tamarin: molecular cloning of the gene encoding the tissue kallikrein

The glandular kallikrein family is composed of structurally related serine proteases. Studies show that the mouse family encompasses at least 14 highly conserved functional genes, but of these only the tissue kallikarein has a human ortholog. In man, the tissue kallikrein display high sequence similarity with prostate specific antigen and human glandular kallikrein 2, suggesting that they evolved after the separation of primates and rodents. A phylogenetic study of the genes encoding glandular kallikreins in species evolutionarily located between rodents and man may reveal interesting details on how the gene family evolved, which in turn could yield information about the function of the proteins. Therefore, we have initiated a study of the glandular kallikreins of the cotton-top tamarin (Saguinus oedipus), a New World Monkey. Here, we report the cloning and nucleotide sequence of one of these, the tissue kallikrein gene. The gene of 4.4 kb is composed of five exons, and the structure is 90% similar to that of the orthologous human gene. It gives rise to a polypeptide of 261 amino acids, including a signal peptide of 17 residues, a pro-piece of 7 residues, and the mature protein of 237 residues with an estimated molecular mass of 26.3 kD. The similarity to the human prostate specific antigen and human glandular kallikrein 2 genes is 73% and 72%, respectively, including introns and flanking regions. The lower similarity to these genes compared with the human tissue kallikrein gene indicates that they, or a progenitor to them, arose in primates prior to the separation of New and Old World monkeys. Genomic Southern blots also show that the cotton-top tamarin genome encompasses at least one more glandular kallikrein gene.