STRUCTURE OF A COVALENTLY CROSS-LINKED FORM OF CORE HISTONES PRESENT IN THE STARFISH SPERM

The post-translational modification of core histones plays an essentia l role in chromatin remodeling processes. We recently reported the occ urrence of a novel histone modification, involving a epsilon-(gamma-gl utamyl)lysine cross-link between a glutamine residue of histone H2B an d a lysine residue of histone H4 in the testis of the starfish, Asteri na pectinifera [Shimizu, T., Hozumi, K., Horiike, S., Nunomura, K., Ik egami, S., Takao, T., and Shimonishi, Y. (1996) Nature 380, 32]. In or der to determine the complete structure of the modified histone hetero dimer, p28 from both testis and sperm was purified. p28 was digested w ith Achromobacter lyticus protease I or Staphylococcus aureus V8 prote ase to give proteolytic fragments that were separated by HPLC. Amino a cid analysis, sequencing, and mass spectrometric analysis of the fragm ents showed that the amino acid sequences of these fragments are ident ical to those of both histones H2B and H4, except for two NH2-terminal peptides obtained by digestion with A. lyticus protease I. One of the peptides, K8, was identical to that reported previously, and the othe r was a here-to-fore unidentified peptide, which was designated K10. A mino acid and positive-ion FAB-MS/MS analyses of K10 showed that it to be a Gly-Glu-Lys Gly-Leu-Gly-Lys-Gly-Gly-Ala-Lys fragment, derived fr om Gly(8)-Lys(10) of histone H2B and Gly(9)-Lys(16) of histone H4. The yields of K8 and K10 were calculated to be 47 and 42%, respectively, expressed as the percent of the total amount of p28 used in the experi ment. Based on these data, the structure of p28 was determined to be a heterodimer, composed of histones H2B and H4, formed through a transg lutaminase-catalyzed acyl transfer reaction between Gln(9) of histone H2B and Lys(5) or Lys(12) of histone H4.