Tektin B1 demonstrates flagellar localization in human sperm.
The human flagellar protein tektin B1 (h-tekB1) in human sperm was cloned, and its sequence and subcellular location were determined. Human sperm proteins were separated by 2-dimensional electrophoresis, and a resolved protein spot of 54 kDa with an isoelectric point (pI) of 5.3 was removed from the gel, trypsinized, and microsequenced by tandem mass spectrometry. The resulting peptides did not match any protein in the (then current) protein databases. Degenerate oligonucleotides based on the microsequences were used with a polymerase chain reaction to amplify a partial cDNA clone from human testis poly(A)(+) mRNA, and subsequently a full-length 1.5-kilobase (kb) clone (GenBank AF054910) was obtained from a testis cDNA library. The open reading frame encoded a 430-amino acid protein with 47% homology to the sea urchin tektin B1. Hybridization of labeled h-tekB1 cDNA to a multiple-tissue Northern blot demonstrated a transcript of 1.7 kb in human testis, and a multiple tissue dot-blot demonstrated high levels of expression in testis, trachea, and lung, intermediate levels in fetal brain and appendix, and low levels in ovary, pituitary, and fetal kidney. Rat polyclonal serum generated against a recombinant h-tekB1 demonstrated 3 h-tekB1 isoforms of pI 5.25, 5.5, and 5.35 at 53.5 kDa on a 2-dimensional Western blot of human sperm proteins. Immunofluorescent studies localized h-tekB1 to the principal piece of human sperm, but the endpiece was unstained.
Wolkowicz
Michael J
MJ
Department of Cell Biology and the Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Naaby-Hansen
Soren
S
Gamble
Angela R
AR
Reddi
P Prabhakara
PP
Flickinger
Charles J
CJ
Herr
John C
JC
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D008868:Microtubule Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D009928:Organ Specificity; D020543:Proteome / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013082:Sperm Tail / Q000378:metabolism*; D013737:Testis / Q000378:metabolism
D018076:DNA, Complementary; D008868:Microtubule Proteins; D015345:Oligonucleotide Probes; D020543:Proteome; D011994:Recombinant Proteins; C044103:tektins