Fore-Lien Huang

Purification and molecular cloning of carp ovarian cystatin

The ovarian fluid of carp consists of many components. Using the antiserum against carp serum, Western blot analysis of ovarian fluid was done in order to distinguish substances synthesized by the ovary from those derived from the serum. Several ovary-specific substances were detected including a protein of 12 kDa (p12), which was purified to homogeneity. Purified p12 displays a single band in SDS-PAGE under nonreducing condition and it can inhibit the enzymatic activity of papain with an apparent inhibition constant of 0.01 nM. The primary structure of p12 was partially determined by Edman degradation and fully elucidated by molecular cloning. A cDNA of 531 bp encoding p12 was obtained. The precursor of p12 has 129 residues, including a signal peptide of 18 residues and a mature protein of 111 residues. The N- and C-terminus of p12 are threonine and methionine, respectively. The p12 shares many common features of the family 2 cystatins of other species, including the similarity of the protein size (in the range of 110 to 120 residues), the presence of 4 cysteine residues and the occurrence of invariant residues throughout the molecule.

The primary structures of growth hormones of three cyprinid species: bighead carp, silver carp and grass carp

The primary structures of growth hormone (GH) of three cyprinid species, bighead carp, silver carp, and grass carp, were determined by a chemical method and/or by molecular cloning. The bighead carp GH was extracted from pituitary tissue by use of an alkaline medium, then successively purified by gel filtration, hydrophobic interaction column chromatography, and reverse-phase high-pressure liquid chromatography. The primary structure of bighead carp GH determined chemically is identical to that deduced from the cDNA nucleotide sequence. By molecular cloning, the primary structures of silver carp and grass carp GHs were also determined. The GHs of these three cyprinid species all contain 188 amino acid residues and their sequences are identical. When four of the five cysteines of cyprinid GHs were arranged to match the same positions of cysteines of other vertebrate GHs, a maximally matched alignment was achieved. Among fishes, GHs are relatively conserved within the same order (82 to 100% identity) but they are more diversified between orders (49 to 68% identity). In further comparison, fish GHs are even more different from tetrapod GHs (37 to 58% identity). Although the primary structures of vertebrate GHs are relatively variable, four homologous sequences, notably one located at the C-terminal, are found.

Purification, characterization, and molecular cloning of gonadotropin subunits of silver carp (Hypophthalmichthys molitrix).

The alpha and beta subunit of silver carp gonadotropin (scGTH-alpha and scGTH-beta) were isolated by high-performance liquid chromatography. Heterogeneity of N-terminal amino acid sequence was observed in scGTH-alpha but not in scGTH-beta. For determining the complete primary structures of scGTH-alpha and scGTH-beta, their cDNAs were cloned. Combining the data of N- and C-terminal sequences determined from proteins and the amino acid sequences deduced from cDNAs, we infer that scGTH-alpha consists of 95 and/or 93 residues and scGTH-beta consists of 115 residues. Both scGTH-alpha and scGTH-beta are glycoprotein. Their carbohydrate content is about 20 g per 100 g protein. The molecular weights of scGTH-alpha and scGTH-beta were calculated to be 12,700 and 15,700 Da, respectively. The amino acid sequences of scGTH-alpha and scGTH-beta are very similar to those of the corresponding subunit of carp GTH, different in only 2 and 4 residues, respectively. In addition, a high extent of homology (70%) was also observed between the alpha subunits of fish and mammalian GTHs. In the case of beta subunit, homology among various species of fish (75 to 98%) is much higher than that between fish and mammal (40%). These data suggest that the alpha subunit is conserved while the beta subunit is diversified during the molecular evolution of vertebrate GTH.

Organization and nucleotide sequence of carp gonadotropin α subunit genes

We have used PCR to amplify and align the sequence of two genes encoding cGTHα. Both genes comprise four exons and three introns. The organization of cGTHα genes is very similar to that of mammalian GTHα genes. However, the cGTHα genes only span a region of 1.2 kb which is much smaller than those mammalian GTHα genes.

A protease inhibitor of the serpin family is a major protein in carp perimeningeal fluid: II. cDNA cloning, sequence analysis, and Escherichia coli expression.

Abstract: A cDNA clone, pCP9, has been isolated from a common carp liver cDNA library by immunoscreening with polyclonal antiserum raised against purified bighead carp α1‐antitrypsin. This clone is 1,396 bp in length and has an open reading frame encoding a protein of 410 amino acid residues. The deduced amino acid sequence shows moderate homology to human α1‐antitrypsin (38%), guinea pig contrapsin (35%), human α1‐antichymotrypsin (34%), and human proteinase C inhibitor (31%), all members of the serine protease inhibitor (serpin) family. To confirm further that the cDNA clone was derived from the authentic carp α1‐antitrypsin gene, the presumptive mature protein of pCP9 was expressed in Escherichia coli. The molecular mass of the recombinant protein matched that predicted from the nucleotide sequence. This recombinant protein, which was recognized by antiserum against native α1‐antitrypsin, was capable of formation of serpin‐enzyme complexes with trypsin, chymotrypsin, and elastase. Therefore, we conclude that the protein encoded by the pCP9 clone is indeed carp α1‐antitrypsin. Expression of α1‐antitrypsin in brain was confirmed by reverse transcription and polymerase chain reaction performed on mRNA derived from both common carp and bighead carp brain.

Molecular cloning of silver carp and bighead carp prolactin

The cDNAs encoding the prolactin of silver carp (scPRL) and bighead carp (bcPRL) have been cloned. Deduced from the nucleotide sequences, both scPRL and bcPRL are composed of 187 amino acid residues. Only one residue is different between scPRL and bcPRL. Homology analysis indicates that scPRL and bcPRL are highly homologous to carp PRL (97%), relatively conserved in relation to PRLs of salmon, trout, and tilapia (64-69%), and diversified from avian and mammalian PRL (30-35%). Similar to PRLs of other species of fish, scPRL and bcPRL lack the first 12 N-terminal residues of avian and mammalian PRLs.

Cloning and expression of carp cathepsin Z: possible involvement in yolk metabolism.

A cDNA encoding cathepsin Z (CTPZ) was cloned from a carp ovarian cDNA library. It is homologous to mammalian CTPZ. The amino acid residues important for protein folding and enzymatic activity of mammalian CTPZ are conserved in carp CTPZ. It is widely expressed in a variety of carp tissues as revealed by Western blot and reverse transcription-polymerase chain reaction. The CTPZ mRNA was transiently accumulated during oocyte maturation. In oocytes, CTPZ is localized in cortical granules and in the cytoplasm surrounding the yolk granules. After fertilization, CTPZ remained associated with the yolk granules while the cortical granular CTPZ was discharged to plasma membrane, perivitelline space, and fertilization envelope. Carp cathepsin Z has proteolytic activity toward vitellogenin that could be inhibited by inhibitors specific for the proteases of papain family. The potential roles of cathepsin Z in carp eggs are discussed.

Separation of subunits of pike eel gonadotropin by hydrophobic interaction chromatography

Subunits of pike eel gonadotropin were dissociated in propionic acid and separated by hydrophobic interaction chromatography on phenyl-Sepharose CL-4B with a high yield (79%). Isolated subunits were homogeneous, as tested by sodium dodecyl sulphate disc electrophoresis and by N-terminal analysis. The biological activities of two subunits, S-I and S-II, were 0% and 2.8% of native molecule, respectively, as assayed by stimulation of androgen production on carp testes. Reassociated molecules restored about 75% activity.

Molecular cloning and sequencing of a carp cDNA encoding mitogen-activated protein kinase kinase.

We have cloned a full-length cDNA encoding the mitogen-activated protein kinase kinase (MKK) from a carp liver cDNA library. The cDNA contains 1970 bp with a single open reading frame encoding a 397 amino acid protein. By comparing with known MKK sequences from other species, carp MKK is 78%, 80%, 76% and 58% identical to rat MKK1, rat MKK2, Xenopus MKK and Drosophila MKK.