Biomedical subjects

H A Kang

Publications and source records attributed to H A Kang.

27 records · Page 2Linked to original sources

PubMed1997

Folding and stability of the Z and S(iiyama) genetic variants of human alpha1-antitrypsin.

Z (Glu342 --> Lys) and S(iiyama) (Ser53 --> Phe) genetic variations of human alpha1-antitrypsin (alpha1-AT) cause a secretion blockage in the hepatocytes, leading to alpha1-AT deficiency in the plasma. Using in vitro folding analysis, we have shown previously that these mutations interfere with the proper folding of polypeptides. To understand the fundamental cause for the secretion defect of the Z and S(iiyama) variants of alpha1-AT, we investigated in vivo folding and stability of these variant alpha1-AT using the secretion system of yeast Saccharomyces cerevisiae. Various thermostable mutations suppressing the folding block of the Z variant in vitro corrected the secretion defect as well as the intracellular degradation in the yeast secretion system. Significantly, the extent of suppression in the secretion defect of Z protein was proportional to the extent of suppression in the folding defect, assuring that the in vivo defect associated with the Z variant is primarily derived from the folding block. In contrast, the folding and secretion efficiency of S(iiyama) was not much improved by the same mutations. In addition, none of the rarely secreted S(iiyama) alpha1-AT carrying the stabilizing mutations for the wild type and Z variant were active. It appears that the major defect in S(iiyama) variant is the loss of stability in contrast to the kinetic block of folding in the Z variant.

Hot Temperature↗

PubMed1996

High-level secretion of human alpha 1-antitrypsin from Saccharomyces cerevisiae using inulinase signal sequence.

The use of a proper signal sequence is one of the major determinants for the efficient secretion of heterologous proteins from yeast. The signal sequence derived from inulinase (INU1A) of Kluyveromyces marxianus was evaluated in directing the secretion of a human glycoprotein, alpha 1-antitrypsin (alpha 1-AT), from Saccharomyces cerevisiae. A yeast expression vector for alpha 1-AT was constructed by placing the coding sequence of human alpha 1-AT fused with the INU1A signal sequence downstream of the GAL10 promoter. S. cerevisiae transformants harboring the expression vector secreted about 70% of the total alpha 1-AT synthesized into the culture media. The intracellularly retained form of alpha 1-AT was mostly unglycosylated, whereas the secreted protein had high mannose-type glycosylation. The fed-batch cultivation of the recombinant yeast achieved a high-cell density, leading to the secretion of biologically active alpha 1-AT up to 75 mg l-1. The secreted protein was purified and subjected to N-terminal sequencing, which confirmed that the secreted alpha 1-AT was processed correctly at the Kex2 cleavage site as expected from the sequence of INU1A signal peptide. The results suggest that the inulinase signal sequence is useful for the high-level secretion of relatively large glycoproteins, such as human alpha 1-AT, from S. cerevisiae.

Amino Acid Sequence↗

PubMed1994

Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae.

Eukaryotic translation initiation factor eIF-5A (formerly eIF-4D) is thought to function in protein synthesis by promoting synthesis of the first peptide bond because it stimulates methionyl-puromycin formation in vitro. eIF-5A is encoded by two genes (TIF51A and TIF51B) in Saccharomyces cerevisiae; the protein and its hypusine modification are essential for cell viability. To analyze the factor's function in vivo, we expressed from the repressible GAL promoter a functional but unstable eIF-5A fusion protein (R-eIF-5A) with an NH2-terminal arginine which is subject to rapid turnover through the NH2-terminal end rule proteolytic pathway. When the conditional mutant strain is shifted from galactose to glucose medium, the rapid disappearance of R-eIF-5A protein occurs within one generation, causing an immediate inhibition of cell growth. However, eIF-5A-depleted cells synthesize protein at about 70% of the wild type rate and exhibit only a slight change in polysome profiles reflecting a subtle defect in a late step of translation initiation. These results suggest that the activity of eIF-5A may not be absolutely essential for general protein synthesis. Rather, eIF-5A may be selectively required for translation of certain mRNAs and/or may be involved in some other aspect of cell metabolism.

Base Sequence↗

PubMed1993

Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine in Saccharomyces cerevisiae.

Translation initiation factor eIF-5A (formerly called eIF-4D) is a small, highly conserved protein in eukaryotic cells that undergoes a unique modification at one of its lysine residues to form hypusine. eIF-5A stimulates in vitro the synthesis of methionyl-puromycin, a model reaction for formation of the first peptide bond. In Saccharomyces cerevisiae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF51B, and each gene gives rise to two hypusinated isoelectric variants, eIF-5Aa (more acidic) and eIF-5Ab (more basic). In order to study the structural and functional differences between the two isoforms, both isoelectric forms were purified from a yeast strain overexpressing TIF51A and were shown to stimulate identically the synthesis of methionyl-puromycin in a heterologous mammalian assay system. Pulse-chase labeling of yeast cells with [35S]methionine showed that the basic form, eIF-5Ab, is a precursor form of the acidic form, eIF-5Aa. Immunoprecipitation of 32P-labeled cell lysates with rabbit antibodies specific for yeast eIF-5A, phosphoprotein phosphatase treatment of eIF-5Aa, and phosphoamino acid analysis demonstrated that eIF-5Aa is generated by phosphorylation of eIF-5Ab on serine. Therefore eIF-5A undergoes two post-translational modifications, hypusination and phosphorylation, where the activity of the factor is dependent on the first but is not influenced in vitro by the second.

Base Sequence↗

PubMed1993

Translation initiation factor eIF-5A expressed from either of two yeast genes or from human cDNA. Functional identity under aerobic and anaerobic conditions.

Translation initiation factor eIF-5A (previously named eIF-4D) is an essential and highly conserved protein in eukaryotic cells that promotes formation of the first peptide bond. One of its lysine residues is post-translationally modified by spermidine to form hypusine, a unique residue required for eIF-5A activity. In Saccharomyces cerevisiae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF51B. The two genes are regulated reciprocally by oxygen, where under aerobic conditions TIF51A is expressed and TIF51B is repressed, and under anaerobic conditions the opposite occurs. In order to study the products of the two genes individually, yeast strains were constructed that express either TIF51A or TIF51B under control of a galactose promoter. Each gene gives rise to two isoelectric variants, eIF-5Aa (more acidic) and eIF-5Ab (more basic), both of which carry the hypusine modification. Expression of either TIF51A or TIF51B promotes growth under both aerobic and anaerobic conditions, indicating that the two gene products function indistinguishably. The human cDNA encoding eIF-5A also was expressed in yeast, and the plasmid shuffle technique was used to demonstrate that the human protein can substitute for the homologous yeast protein in vivo. These results indicate that human and yeast eIF-5A are not only conserved at the sequence level but are functionally interchangeable in vivo.

Aerobiosis↗

PubMed1992

The two genes encoding protein synthesis initiation factor eIF-5A in Saccharomyces cerevisiae are members of a duplicated gene cluster.

Translation initiation factor eIF-5A is an abundant protein in which a lysine residue is modified by spermidine to form the amino acid derivative, hypusine. The factor is encoded by two genes in Saccharomyces cerevisiae, called TIF51A and TIF51B, which are regulated reciprocally by oxygen and by heme. TIF51B, also called ANB1, is located on chromosome X in a region called COR. We physically mapped TIF51A and its associated serine tRNA2 gene by the method of chromosome fragmentation and pulsed-field gel electrophoresis. TIF51A maps 90 kb from the left end of chromosome V in a region called ARC. The COR and ARC regions contain CYC1 and CYC7, respectively, and appear to be duplications carrying numerous related genes. The arrangements of related genes in the two regions are incompatible with a duplication mechanism involving a circular intermediate.

Electrophoresis, Gel, Pulsed-Field↗

PubMed1991

Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae.

Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIF51A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca. 63% identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of the par locus on the growth rate and structural stability of recombinant cells.

A decreased growth rate of recombinant cells is observed when a cloned gene protein encoded in a multicopy plasmid is induced from a strong promoter. This negative effect on the cell growth rate may be the consequence of alternate use or reallocation of energy, precursor metabolites, and protein synthesizing machinery. In order to analyze the causes for this adverse effect, we have studied how genetic elements present in multicopy plasmids affect the growth of recombinant Escherichia coli (K12 delta H1 delta trpEA). Turning on of the PL promoter, whether or not protein-coding sequences were present downstream of the promoter, decreased the growth of the recombinant cells by 15-50%. This result suggests that the essential factor(s) for cell growth may be sequestered by the genetic elements present in the plasmids and thus may cause the decreased growth. The negative effect of the de-repressed PL promoters on cell growth was partially reversed when the par sequence from pSC101 was inserted into the same plasmid. In each case when the plasmid carried only the PL promoter, the PL promoter followed by the N-terminal part of the protein-coding region, or the PL promoter followed by complete sequences encoding a protein, the par sequence exhibited a positive effect on the growth rate of the recombinant cells, which usually grow at a slower rate than the host cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMedDate not supplied

The comparative molecular study between Bombycidae and Saturniidae based on mtDNA RFLP and cytochrome oxidase I gene sequences: implication for molecular evolution.

The phylogenetic relationships between Bombyx mori and Bombyx mandarina species of Bombycidae, and Antheraea yamamai and Antheraea pernyi species of Saturniidae were investigated based on mtDNA RFLP and cytochrome oxidase I gene. The sizes of the mtDNA of all the species were estimated at approximately 16 kbp +/- 500 bp by total length of all the restricted fragments and no variation in size was recognized. Of the fourteen different restriction endonucleases used, BamHI, HindIII, PstI, EcoRI and XbaI showed RFLP. Among these, only HindIII showed RFLP between B. mori and B. mandarina. A comparative analysis of sequences was also conducted with the mitochondrial cytochrome oxidase I genes of each species. The results indicated that B. mori shared a 97%, 85% and 87% sequence identity with B. mandarina, A. yamamai and A. pernyi, respectively. B. mandarina shared a 87% and 88% sequence identity with A. yamamai and A. pernyi, respectively. A. yamamai shared 92% sequence identity with A. pernyi. The results of the phylogenetic analysis exhibited monophyly and confidence limits of more than 99% in all trees for both Bombycidae and Saturniidae.

Animals↗