Objective: To explore the binding domain of hydroxypyruvate isomerase homologues (HYI) in the interaction with protein P311 in hypertrophic scar fibroblasts (Fb).
Methods: (1) P 311 was amplified by PCR using plasmid pMD18-T-P 311 as template. The total RNA of hypertrophic scar Fb was extracted by Trizol to amplify HYI with RT-PCR. Recombinant vectors pGADT7-P 311 and pGBKT7-HYI were constructed by double-enzyme digestion, and they were verified by PCR and sequencing. The secondary structure of protein HYI was analyzed with software Prot Seale and HNN. Fragments of HYI-1 (1-447 bp), HYI-2 (247-447 bp), HYI-3 (1-279 bp), and HYI-4 (247-654 bp) were amplified based on the result of software analysis. And then the recombinant vectors pGBKT7-HYI-1, 2, 3, and 4 were constructed by double-enzyme digestion and verified by PCR and sequencing. (2) AH109 yeast cells were transformed into competent cells by lithium acetate method and divided into 7 groups roughly in the same amount, including HYI full length, HYI-1, HYI-2, HYI-3, and HYI-4 hybrid groups, positive control group, and negative control group. Cells in the first five groups were respectively transformed with recombinant vector pGBKT7-HYI full length, pGBKT7-HYI-1, pGBKT7-HYI-2, pGBKT7-HYI-3, pGBKT7-HYI-4 and recombinant vector pGADT7-P 311, and that in the rest two groups were transformed with recombinant vectors pGBKT7-53 and pGADT7-RecT, pGADT7-RecT and pGBKT7-Lam by polyethyleneglycol/lithium acetate method. Immediately after transformation, a part of the transformed cells in each group was spread onto the medium lacking leucine, tryptophan, adenine, and histidine (briefly called four-factor lacking medium), and another portion of the cells was spread onto the medium lacking leucine and tryptophan (briefly called two-factor lacking medium). After 3 to 6 days' culture, the growth of yeast was observed, and the expression of β-galactosidase of yeast was detected by color reaction with 5-bromo-4-chloro-indolyl-β-D-galactopyranoside.
Results: (1) Cloned P 311 and the reported P 311 (GenBank ID hsu36189) had the same sequence. The A base at 496 bp in reported HYI (GenBank ID AY775560) was replaced by G base as found in cloned HYI. It was verified that the insert segment of each recombinant vector was correct. (2) Among those 216 amino acids which composed the protein HYI, 101 amino acids might form α helices, 90 amino acids might form random coils, 25 amino acids might form extended-chains as revealed in the simulated structure analysis by computer. (3) Cloned segments HYI-1, 2, 3, 4 showed expected lengths. It was verified that the insert segment of each recombinant vector was correct. (4) Except for strains in negative control group which did not show growth on four-factor lacking medium, all strains in other groups grew on both kinds of media, and growth of colonies was less in HYI-2 (with the fewest number of α helices) and HYI-3 hybrid groups. (5) Positive expression of β-galactosidase was observed in strains of all groups growing on four-factor lacking medium except for the HYI-2 hybrid group. No expression of β-galactosidase was observed in strains of negative control group which grew on two-factor lacking medium.
Conclusions: Protein HYI may closely bind with protein P311 by α helix, which plays an important role in fibroblast-to-myofibroblast transdifferentiation in hypertrophic scar.