Differential O-glycosylation of a conserved domain expressed in murine and human ZP3.
Murine sperm initiate fertilization by binding to the zona pellucida (mZP), the specialized extracellular matrix of their homologous eggs. O-Glycans occupying two highly conserved vicinal glycosylation sites (Ser-332 and Ser-334) on the mZP glycoprotein designated mZP3 were previously implicated in this interaction. However, recent biophysical analyses confirm that neither site is occupied, implying that an alternate O-glycosylation domain may be operational in native mZP3. Since human ZP3 (huZP3) can substitute for mZP3 in rescue mice to mediate sperm binding, the site specificity of O-glycosylation in both native mZP3 and huZP3 was analyzed using ultrasensitive mass spectrometric techniques. Two O-glycosylation sites in native mZP3, one at Thr-155 and the other within the glycopeptide at positions 161-168 (ATVSSEEK), are conserved in huZP3 derived from transgenic mice. Thus, there is a specific O-glycosylation domain within native mZP3 expressing two closely spaced O-glycans that is very well conserved in an evolutionarily related glycoprotein. In native mZP3, core 2 O-glycans predominate at both sites. However, in huZP3 derived from rescue mice, the O-glycans associated with Thr-156 (analogous to Thr-155 in mZP3) are exclusively core 1 and related Tn sequences, whereas core 2 O-glycans predominate at the other conserved site. This unique restriction of O-glycan expression suggests that sequence differences in the conserved O-glycosylation domains of mZP3 and huZP3 affect the ability of core 2 N-acetylglucosaminyltransferase(s) to extend the core 1 sequence. However, this difference in O-glycosylation at Thr-156 does not affect the fertility or the sperm binding phenotype of eggs derived from female huZP3 rescue mice.
Chalabi
Sara
S
Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.
Panico
Maria
M
Sutton-Smith
Mark
M
Haslam
Stuart M
SM
Patankar
Manish S
MS
Lattanzio
Frank A
FA
Morris
Howard R
HR
Clark
Gary F
GF
Dell
Anne
A
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D017124:Conserved Sequence / Q000235:genetics; D004527:Egg Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010053:Ovary / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse; D013912:Threonine