Keratin 9 is a component of the perinuclear ring of the manchette of rat spermatids

Previous work in our laboratory has shown that a 62- to 64-kDa protein was a major component of the perinuclear ring of manchettes fractionated from r at spermatids. Mass spectrometry analysis of this protein indicated the pre sence of a glycine-rich domain homologous to human keratin 9 (K9). Several antibodies to K9, raised against synthetic peptides of human K9, recognized the 64- to 62-kDa protein in the perinuclear ring of the manchette as well as in keratinocytes of the suprabasal layer of the rat and human footpad/s ole epidermis in both immunoblotting and immunocytochemical experiments. Ba sed on these data, human-derived K9 primers were used to clone rat K9 cDNA from epidermis by RT-PCR. Rat-specific K9 primers were then used to perform a two-step (nested) PCR to amplify the K9-specific rat testicular RNA and to obtain cDNA to demonstrate K9 gene expression in rat testis. The deduced amino acid sequence of rat K9 cDNA contains 618 amino acids with an estima ted molecular mass of 63,020 Da, in agreement with that obtained by electro phoretic fractionation of rat manchette and epidermis footpad proteins. The deduced protein structure correlates with the recognizable pattern of kera tins: a rod domain of 304 amino acids with well-conserved initiation and te rmination sequences (MQNLNSRLASY and EIETYRKLLEG, respectively), flanked by glycine/serine-rich head and tail domains of 141 and 173 amino acids, resp ectively. A high content of phenylalanine was detected in the head domain a nd a repetitive motif (SGGSYGGGS) in the tail domain. A comparison with hum an keratin 9 showed an overall nucleotide and amino acid similarity of 75%. An increased level of K9 transcripts was detected in a cDNA library prepar ed from fractionated round spermatids. Results of this study show that rat testis expresses K9 and that this protein is a component the perinuclear ri ng of the manchette of rat spermatids. (C) 2000 Academic Press.