Differential O-glycosylation of a conserved domain expressed in murine and human ZP3

Biochemistry. 2006 Jan 17;45(2):637-47. doi: 10.1021/bi0512804.


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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Carbohydrate Sequence
  • Conserved Sequence* / genetics
  • Egg Proteins / biosynthesis
  • Egg Proteins / genetics
  • Egg Proteins / metabolism*
  • Female
  • Glycosylation
  • Humans
  • Membrane Glycoproteins / biosynthesis
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Mice, Transgenic
  • Molecular Sequence Data
  • Ovary / metabolism
  • Protein Structure, Tertiary / genetics
  • Receptors, Cell Surface / biosynthesis
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism*
  • Spectrometry, Mass, Electrospray Ionization
  • Threonine / genetics
  • Threonine / metabolism
  • Zona Pellucida Glycoproteins


  • Egg Proteins
  • Membrane Glycoproteins
  • Receptors, Cell Surface
  • ZP3 protein, human
  • Zona Pellucida Glycoproteins
  • Zp3 protein, mouse
  • Threonine