ИСТИНА

Human glyceraldehyde-3-phosphate dehydrogenase catalyzes one of the glycolytic reactions and exists in two homologous isoenzyme forms: GAPD, a well-studied protein found in all somatic cells, and GAPDS, which is expressed solely in testes. GAPDS supplies energy required for the movement of spermatozoa and is tightly bound to the sperm tail cytoskeleton. Actually, there is only little data on GAPDS properties. GAPDS stability towards denaturation was found to be enhanced as compared to GAPD that may be an adaptation to the absence of protein expression in spermatozoa. In the present study we investigated the structural features of GAPDS accountable for its enhanced stability. At the first step, the GAPD and GAPDS sequences and x-ray structures were computationally analyzed. The following specific features that may affect stability were revealed as a result: (1) 7 additional proline residues in GAPDS, with 6 of them meeting the “proline rule”; (2) 8 additional glycine residues in GAPD; and (3) 2 additional semi-buried interdomain salt bridges in GAPDS. At the second step, the molecular modeling of a series of GAPD and GAPDS mutant variants was carried out and it was shown that all of the features listed above except one of the salt bridges could be somewhat accountable for the GAPDS enhanced stability. At the third step, we used the site-directed mutagenesis approach to introduce mutations to the recombinant GAPDS protein which were assumed to lessen its stability. The subsequent experimental assessment of the stability of the obtained mutant proteins proved our assumptions. The work was supported by the Russian Foundation for Basic Researches (grants 12-04-91330-NNIO_a and 12-04-01164-а).