Dataset Information

Molecular cloning reveals isoforms of bovine alpha 1-antichymotrypsin.

ABSTRACT: Comparison of bovine alpha 1-antichymotrypsin (ACT) protease inhibitor with that in human was achieved by cloning a nearly full-length bovine ACT cDNA of 1.5 kb, obtained by screening a bovine liver cDNA library with the human liver ACT cDNA. The deduced primary sequence indicated that the 1456-bp bovine ACT cDNA encodes a protein of 416 amino acids that contains the predicted full-length ACT with a 26-residue NH2-terminal signal sequence. Overall, the primary sequence of bovine ACT possesses a high degree of homology (55%) with human ACT; both bovine and human ACTs share common sequences in the reactive-site domains. Importantly, the reactive site of bovine ACT possesses serine as the predicted P1 position (residue at the NH2-terminal side of the cleaved peptide bond) of the reactive site, whereas human ACT contains leucine in the P1 position. Interestingly, further evidence for heterogeneity in P1 residues was provided by a second partial 0.9-kb bovine liver ACT cDNA clone (pHHK11) that contains isoleucine as P1 residue and shares only partial homology (68%) with the deduced primary sequence of the full-length bovine liver ACT cDNA clone (pHHK12). These findings suggest that isoforms of ACT in bovine liver vary in reactive-site P1 residues; the P1 position of the reactive site is often involved in protease inhibitor specificity. Consistent with the hypothesis of ACT isoforms was the demonstration of multiple copies of the bovine ACT gene by genomic blots.

SUBMITTER: Hwang SR

PROVIDER: S-EPMC44856 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Molecular cloning reveals isoforms of bovine alpha 1-antichymotrypsin.

Hwang S R SR Kohn A B AB Hook V Y VY

Proceedings of the National Academy of Sciences of the United States of America 19940901 20

Comparison of bovine alpha 1-antichymotrypsin (ACT) protease inhibitor with that in human was achieved by cloning a nearly full-length bovine ACT cDNA of 1.5 kb, obtained by screening a bovine liver cDNA library with the human liver ACT cDNA. The deduced primary sequence indicated that the 1456-bp bovine ACT cDNA encodes a protein of 416 amino acids that contains the predicted full-length ACT with a 26-residue NH2-terminal signal sequence. Overall, the primary sequence of bovine ACT possesses a ...[more]

PMID: 7937809

Similar Datasets

Project description:Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

Project description:Monoclonal antibodies to fibrillin 1 (MP340), a component of elastin-associated microfibrils, were used to screen cDNA libraries made from bovine nuchal ligament mRNA. One of the selected clones (cL9; 1.2 kb) hybridized on Northern (RNA) blotting with nuchal ligament mRNA to two abundant mRNAs of 9.0 and 7.5 kb, which were clearly distinct from fibrillin mRNA (10 kb). Further library screening and later reverse transcription PCR by the rapid amplification of cDNA ends (RACE) technique resulted in the isolation of additional overlapping cDNAs corresponding to about 6.7 kb of the mRNA. The encoded protein exhibited sequence similarity of around 80% with a recently identified human protein named latent transforming growth factor beta 1 (TGF-beta 1)-binding protein 2 (LTBP-2), indicating that the new protein was bovine LTBP-2. This was confirmed by the specific localization of bovine LTBP-2 cDNA probes to human chromosome 14q24.3, which is the locus of the human LTBP-2 gene. The domain structure of bovine LTBP-2 is very similar to that of the human LTBP-2, containing 20 examples of 6-cysteine epidermal growth factor-like repeats, 16 of which have the consensus sequence for calcium binding, together with 4 examples of 8-cysteine motifs characteristic of fibrillins and LTBP-1. A 4-cysteine sequence which is unique to bovine LTBP-2 and which has similarity to the 8-cysteine motifs was also present. Antibodies raised to two unique bovine LTBP-2 peptides specifically localized in tissue sections to the elastin-associated microfibrils, indicating that LTBP-2 is closely associated with these structures. Immunoblotting experiments identified putative LTBP-2 isoforms as a 260-kDa species released into the medium by cultured elastic tissue cells and as larger 290- and 310-kDa species in tissue extracts. A major proportion of tissue-derived LTBP-2 required treatment with 6 M guanidine for solubilization, indicating that the protein was strongly bound to the microfibrils. Most of the guanidine-solubilized LTBP-2 appeared to be monomeric, indicating that it was not involved in disulfide-bonded aggregation either with itself or with latent TGF-beta. Additional LTBP-2 was resistant to solubilization with 6 M guanidine but was readily extracted with a reductive saline solution. This treatment is relatively specific for solubilization of microfibrillar constituents including fibrillin 1 and microfibril-associated glycoprotein. Therefore, it can be inferred that some LTBP-2 is bound covalently to the microfibrils by reducible disulfide linkages. The evidence suggests that LTBP-2 has a direct role in elastic fiber structure and assembly which may be independent of its growth factor-binding properties. Thus, LTBP-2 appears to share functional characteristics with both LTBP-1 and fibrillins.

Dataset Information

Molecular cloning reveals isoforms of bovine alpha 1-antichymotrypsin.

Publications

Molecular cloning reveals isoforms of bovine alpha 1-antichymotrypsin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets