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 rat spermatids. Mass spectrometry analysis of this protein indicated the presence 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/sole epidermis in both immunoblotting and immunocytochemical experiments. Based 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 estimated molecular mass of 63,020 Da, in agreement with that obtained by electrophoretic fractionation of rat manchette and epidermis footpad proteins. The deduced protein structure correlates with the recognizable pattern of keratins: a rod domain of 304 amino acids with well-conserved initiation and termination sequences (MQNLNSRLASY and EIETYRKLLEG, respectively), flanked by glycine/serine-rich head and tail domains of 141 and 173 amino acids, respectively. A high content of phenylalanine was detected in the head domain and a repetitive motif (SGGSYGGGS) in the tail domain. A comparison with human keratin 9 showed an overall nucleotide and amino acid similarity of 75%. An increased level of K9 transcripts was detected in a cDNA library prepared from fractionated round spermatids. Results of this study show that rat testis expresses K9 and that this protein is a component the perinuclear ring of the manchette of rat spermatids.
Mochida
K
K
Department of Cell Biology and Anatomical Sciences, The City University of New York Medical School, New York, New York 10031, USA.
Rivkin
E
E
Gil
M
M
Kierszenbaum
A L
AL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004817:Epidermis / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007633:Keratins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013087:Spermatids / Q000378:metabolism*; D013737:Testis / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D007633:Keratins