Identification of a novel GPI-anchored CRISP glycoprotein, MAK248, located on the posterior head and equatorial segment of cynomolgus macaque sperm

A. I. Yudin, M. W. Li, K. R. Robertson, Theodore L Tollner, G. N. Cherr, J. W. Overstreet

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

To identify a sperm-surface component that is highly antigenic, we immunized female cynomolgus macaques with glycosylphosphatidylinositol (GPI)-anchored sperm surface proteins that were released following treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Five different adjuvants were used in combination with the PI-PLC-released proteins, and three of these proteins (24, 48, and 53 kDa) were shown to be potent antigens for immunization of female monkeys. The 53 kDa protein was found to be a surface coating protein and not a GPI-anchored protein. Polyclonal antibodies to the 24 kDa protein and the 48 kDa protein were produced in rabbits. The two antibodies recognized both proteins on Western blots. The same rabbit antibodies recognized 28, 18, and 10 kDa bands on a Western blot of chemically reduced PI-PLC-released proteins, suggesting that the 48 kDa protein is a dimer of the 24 kDa protein, which we refer to as MAK248. Rabbit polyclonal antibodies developed to reduced fragments of the 24 kDa protein showed that the 18 and 10 kDa bands are proteolytic peptide fragments of the 24 kDa protein. Screening of tissues from male macaques showed that MAK248 is expressed only in the epididymis. Microsequencing of two proteolytic fragments of the 18 kDa component showed 100% amino acid homology to a 233 deduced amino acid sequence previously identified in human testes genome. Antibodies to MAK248 recognized a 24 kDa protein released from human sperm exposed to PI-PLC. Antibodies to MAK248 recognized the equatorial segment and posterior head regions of capacitated cynomolgus macaque sperm. Structural analysis suggests that MAK248 is a novel CRISP protein and a member of the CAP (CRISP, Ag 5, PR-1) family of proteins. Based on amino acid sequence homology, it is possible that MAK248 functions as a protease inhibitor.

Original languageEnglish (US)
Pages (from-to)488-499
Number of pages12
JournalMolecular Reproduction and Development
Volume63
Issue number4
DOIs
StatePublished - Dec 1 2002

Fingerprint

Glycosylphosphatidylinositols
Macaca
Spermatozoa
Glycoproteins
Head
Proteins
Phosphoinositide Phospholipase C
Antibodies
Rabbits
Membrane Proteins
Western Blotting
Amino Acid Sequence Homology
Peptide Fragments
Epididymis
Human Genome
Protease Inhibitors

Keywords

  • CRISP
  • Epididymis
  • GPI-anchored
  • MAK248
  • Sperm

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology
  • Cell Biology

Cite this

Identification of a novel GPI-anchored CRISP glycoprotein, MAK248, located on the posterior head and equatorial segment of cynomolgus macaque sperm. / Yudin, A. I.; Li, M. W.; Robertson, K. R.; Tollner, Theodore L; Cherr, G. N.; Overstreet, J. W.

In: Molecular Reproduction and Development, Vol. 63, No. 4, 01.12.2002, p. 488-499.

Research output: Contribution to journalArticle

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abstract = "To identify a sperm-surface component that is highly antigenic, we immunized female cynomolgus macaques with glycosylphosphatidylinositol (GPI)-anchored sperm surface proteins that were released following treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). Five different adjuvants were used in combination with the PI-PLC-released proteins, and three of these proteins (24, 48, and 53 kDa) were shown to be potent antigens for immunization of female monkeys. The 53 kDa protein was found to be a surface coating protein and not a GPI-anchored protein. Polyclonal antibodies to the 24 kDa protein and the 48 kDa protein were produced in rabbits. The two antibodies recognized both proteins on Western blots. The same rabbit antibodies recognized 28, 18, and 10 kDa bands on a Western blot of chemically reduced PI-PLC-released proteins, suggesting that the 48 kDa protein is a dimer of the 24 kDa protein, which we refer to as MAK248. Rabbit polyclonal antibodies developed to reduced fragments of the 24 kDa protein showed that the 18 and 10 kDa bands are proteolytic peptide fragments of the 24 kDa protein. Screening of tissues from male macaques showed that MAK248 is expressed only in the epididymis. Microsequencing of two proteolytic fragments of the 18 kDa component showed 100{\%} amino acid homology to a 233 deduced amino acid sequence previously identified in human testes genome. Antibodies to MAK248 recognized a 24 kDa protein released from human sperm exposed to PI-PLC. Antibodies to MAK248 recognized the equatorial segment and posterior head regions of capacitated cynomolgus macaque sperm. Structural analysis suggests that MAK248 is a novel CRISP protein and a member of the CAP (CRISP, Ag 5, PR-1) family of proteins. Based on amino acid sequence homology, it is possible that MAK248 functions as a protease inhibitor.",
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