Identification and characterization of GH receptor and serum GH-binding protein in Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon, a kind of cartilage ganoid, has a history of over one billion years and it is called the living fossil of aquatic biology since it keeps some evolutionary trace. Here, we characterized the growth hormone receptor (GHR) and serum growth hormone binding protein (GHBP) of Chinese sturgeon. It was shown that GHR was expressed in various tissues, mainly in hepatic, kidney and intestine tissues. GHR on the hepatic membrane has high and specific affinity for bream GH (brGH) and Scatchard analysis of the binding data showed a single class of high affinity binding site with an association constant Ka of 3.1x10(9) M(-1). A specific band around 94 kD was detected by SDS-PAGE in cross-linking studies of membrane receptors. After incubation of Chinese sturgeon serum with 125I-brGH, a 125I-brGH-GHBP complex was identified by Sephadex G-75, indicating that in the serum exists GHBP specially binding to brGH.

Construction of secretory expression system suitable to express glucagon under the control of PL promoter.

It was reported that PL promoter and alkaline phosphatase (phoA) signal peptide were used to construct secretory expression plasmid suitable to express glucagon and [Des-His1] glucagon in E. coli BL21 herein. Expression studies showed these two peptides could be expressed and secreted into the culture medium. The expression yield of recombinant glucagon reached 3.46 mg/L/OD600 unit of cells in shake flask. The yield of [Des-His1] glucagon was found to be higher than that of glucagon. In addition, some factors involved in secretion were studied too.

Functional expression of guinea pig growth hormone receptor and its mutants in mammalian cells.

The cDNA of guinea pig (Cavia porcellus) growth hormone receptor (gpGHR) was cloned using RT-PCR in our laboratory. By sequence alignment, substitutions of amino acids conserved in other mammalian GHRs were found. For example, histidine-168 and tyrosine-332 equivalent to positions 170 and 333 in other mammalian GHRs, which were considered to be necessary for the dimerization of GHR and the specific GH-stimulated functions respectively, were replaced by tyrosine and serine in gpGHR. Here, we report the functional expression of gpGHR and its mutants, gpGHRY168H and gpGHRS332Y, in COS-7 cells and/or Chinese hamster ovary (CHO) cells. It was shown that the COS-7 cells transfected with pcDNA3-gpGHR possessed high affinity to bovine GH [K(a) = 1.3 x10(9) (mol/L)(-1)] and a protein band with molecular weight around 92 kD was detected by anti-mouse GHR monoclonal antibody (mAb263). When CHO cells were transfected with the expression vectors, pcDNA3-gpGHR, pcDNA3-gpGHRY168H and pcDNA3-gpGHRS332Y, the gpGHR and its mutants were expressed and the ligand binding, phosphorylation of JAK2, protein synthesis, and lipogenesis were studied. It was found that the mutation of serine to tyrosine at position 332 greatly increased the GH-stimulated protein synthesis and the phosphorylation of JAK2, while the mutation of tyrosine to histidine at position 168 increased the protein synthesis and decreased the phosphorylation of JAK2 only weakly. However, both mutations decreased the GH-stimulated lipogenesis. Thus, our study provides the experimental evidence that gpGHR may mediate the metabolic actions of GH and the substitutions of some conserved amino acids in gpGHR result in the changes of post-binding signaling.

[Secretory Expression of the Superactive [Lys(17,18),Glu(21)]-Glucagon in E. coli].

One of the most important findings in structure-function studies on glucagon by means of chemical synthesis is the discovery that [Lys(17,18),Glu(21)]-glucagon had higher biological activity than native glucagon. This mutant of glucagon was called superactive glucagon (SA-glucagon). In the present work, the possibility to obtain SA-glucagon by means of genetic engineering was studied. The gene of SA-glucagon (SAG) was obtained by PCR from a constructed recombinant glucagon plasmid, pAGluT. A secretory expression vector harboring SAG, pBLSG7, containing P(L) promoter and the gene of phoA signal peptide was constructed. In expression studies after transformation of pBLSG7 into E. coli BL21, it was found that the expression yield of SA-glucagon reached 3.65 mg/L(A(600)=1), about 19.5% of total proteins in the culture medium under shaken flask conditions. In addition, the influence of induction temperature and of E. coli strain on the expression yield of SA-glucagon was also studied.

Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin.

Blood glucose lowering assay proved that [B16Ala]insulin and [B26Ala]insulin exhibit potency of acute blood glucose lowering in normal pigs, which demonstrates that they are fast-acting insulin. Single-chain precursor of [B16Ala]insulin and [B26Ala]insulin is [B16Ala]PIP and [B26Ala]PIP, respectively, which are suitable for gene expression. Secretory expression level of the precursors in methylotrophic yeast Pichia pastoris was quite high, 650 mg/L and 130 mg/L, respectively. In vivo biological assay showed that the two fast-acting insulins have full or nearly full biological activity. So both [B16Ala]insulin and [B26Ala]insulin can be well developed as fast-acting insulin for clinic use.

Effects of Divalent Cations and Disulfide Bond Reducing Agents on Specific Binding of Growth Hormone to Liver Membrane Receptors from Snakehead Fish (Ophiocephalus argus, Cantor).

Divalent cations, Ca(2 ), Mg(2 ) and Mn(2 ) enhance the binding of bream growth hormone (brGH) to snakehead fish liver membrane, and their optimum concentration was found to be 8 12 mmol/L, at which Ca(2 ), Mg(2 ) and Mn(2 ) could increase, respectively, the specific binding to 230%, 180%, and 200%, compared with the binding in the absence of ions. The Eadie-Scatchard plot was used for the dynamic analysis of the Ca(2 ) binding site. A low affinity Ca(2 ) binding site was found in the GH-receptor complex with K(m)=0.384 mmol/L, and the affinity constant (K(a)) was increased from 1.045x10(9) L.mol(-1) to 1.295x10(9) L.mol(-1) by the addition of 10 mmol/L CaCl(2). The effects of disulfide bond reducing agents, DTT and ME, on (125)I-brGH binding to growth hormone receptor (GHR) on snakehead fish liver memebrane were also analyzed. The addition of 0.1 20 mmol/L DTT or 0.01% 1% ME to the radioreceptor assay system caused a significant dose dependent increase in the specific binding for (125)I-brGH. In the presence of 0.8 mmol/L DTT or 0.08% ME, the specific binding of (125)I-brGH was increased from 10.2% to 15.5% and 13.2% respectively, and the affinity constant was also increased from 1.265x10(9) L.mol(-1) to 2.185x10(9) L.mol(-1) and 1.625x10(9) L.mol(-1), respectively but no changes in the binding capacity were observed. Further studies showed that the effects of reductants on the specific binding of brGH were due in part to the ligand itself and in part to GHR. In addition, it was observed that one of the three disulfide bonds of brGH could be reduced by 0.8 mmol/L DTT.

cDNA Cloning of the Guinea Pig Growth Hormone Receptor.

cDNA cloning and 1 899 bp sequence of growth hormone receptor (GHR) from guinea pig liver are described. The guinea pig GHR consists of 610 amino acids. The structural feature and homology comparison of guinea pig GHR are also reported.

cDNA Cloning of the Cytoplasmic Domain of Growth Hormone Receptor from guinea Pig and Homology Comparison.

The evolutionary position of guinea pig has not been unequivocally clarified, and it appears to show abnormalities in GH response. The cDNA of GHR cytoplasmic domain from guinea pig was cloned and sequenced. By homology comparison, it was found that the sequence we obtained was different markedly from that of rat or mouse. The results provided not only the molecular biology evidence for clarifying the evolutionary position of guinea pig, but also a basis to determine the complete cDNA sequence of GHR from guinea pig and to understand its cytoplasmic signal transduction.

Preparation and Biological Activity of [B(1)Ala, B(2)Ala, B(3)Lys]-Insulin.

This paper describes the preparation and biological activity of an insulin analogue in which the B1-3 sequence (Phe-Val-Asn) of insulin is substituted by Ala-Ala-Lys. [B(1)Ala, B(2)Ala, B(3)Lys]-Insulin retains full in vivo activity and receptor binding activity as insulin, but its lipogenesis activity and immunoactivity are 70 % and 0.88 % of those of insulin respectively. The possible contribution of the N-terminus of B-chain of insulin to the structure and function of insulin is discussed.

Protein Engineering of Insulin: [B9 Glutamic Acid] Human Insulin.

B9Ser of insulin B chain was substituted by Glu using site-directed mutagenesis to obtain a fast-acting insulin-[B9Glu] human insulin. The receptor binding capacity and in vivo biological activity of [B9Glu] human insulin are 21% and 40% as those of porcine insulin respectively.

The Receptor Binding and Biological Activity of [B1-Ala, B2-Ala]-insulin.

The receptor binding properties with human placental membrane (HPM) and the in vitro biological activity of an insulin analogue, [B1-Ala, B2-Ala]-insulin were investigated in detail and compared with those of insulin. It was found that the binding of (125)I-[B1-Ala, B2-Ala]-insulin and (125)I-insulin to HPM was time dependent reaching equilibrium after 6min at 37 degrees in the presence of bacitracin with an equilibrium maximum binding of 6.44 fmol/mg protein for [B1-Ala, B2-Ala]-insulin and 3.47 fmol/mg protein for insulin The half time (T1/2) to reach equilibrium was 19 seconds for [B1-Ala, B2-Ala]-insulin and 25 seconds for insulin. [B1-Ala, B2-Ala]-insulin and insulin competed with specific (125)I-[B1-Ala, B2-Ala]-insulin in a dose-dependent manner. From IC(50), the receptor binding activity of [B1-Ala, B2-Ala]-insulin was 139.6% compared with that of insulin. Scatchard analysis revealed that the receptor association constants of [B1-Ala, B2-Ala]-insulin in HPM at 4 degrees were 5.88x10(8)L/mol and 7.63x10(5)L/mol respectively, while those of insulin were 4.83x10(8)L/mol and 3.39x10(5)L/mol respectively. The in vitro activity of [B1-Ala, B2-Ala]-insulin was 130% of that of insulin in terms of lipogenesis in rat adipocytes.