Oviduct-specific glycoprotein, OVGP, is a secreted protein found exclusively in the oviduct and is subject to several signaling events, including hormonal induction.1 It is believed that this protein plays an important role in early embryogenesis, possibly acting positively on sperm motility and sperm-egg interaction, as well as a having possible role preventing polyspermy.1,2 OVGP1 is also heavily modified through glycosylation. There is evidence that the different modifications play an important role in protein solubility and interaction with sperm. While at least four separate glycosylation sites have been identified on OVGP1, the specific identities of these glycan structures have not been individually analyzed. It is believed that these sites may display highly diversified structures, possibly as a mechanism to identify species, prevent proteolysis, or stabilize protein-protein interactions.1,2 A greater understanding of the specific glycan structures attached to the sites on OVGP1 could give valuable insight into the overall function of these modifications.
Further study of these sites has been hampered partly by the lack of materials to analyze. The narrow tissue specificity and relatively low amounts of protein present make the analysis of the glycan structures difficult. The first hurdle to clear with the study of this protein is creating a recombinant system for higher expression with intact modifications. In Yang et al.,3 the researchers demonstrate that recombinant human oviduct-specific glycoprotein (rhOVGP1) can be produced in relatively high amounts using a human embryonic kidney cell line (HEK293). Moreover, the rhOVGP1 is also glycosylated. Glycosylation was first demonstrated through electrophoretic mobility after treatment with inhibitors GalNAca-Bn and tunicamycin. The enzyme neuraminidase also affected the mobility of rhOVGP1, indicating the modifications were sialyated. These results are consistent with the wild-type protein.
The next task of the researchers was to determine the exact chemical nature of the modifications through mass spectrometry and enzymology and to determine if these modifications do have an effect on the bioactivity of this protein. Using a linear ion trap Fourier transform mass spectrometer (LTQ-FT, Thermo Scientific) on PNGase F treated rhOVGP1 revealed the presence of nine different O-linked glycan chains ranging from 2 to 6 residues in length and a huge variety of multi-branched N-linked glycosylations detected by MS. In all, more than 20 different branched N-linked structures were mapped, including mannose chains and bi-, tri-, and tetra-antennary complex nonbisected and bisected chains.3 The composition of each modification was determined by molecular weight, isotopic peak distribution, and MS/MS fragmentation. Now that the modifications were mapped and thoroughly characterized by mass spectrometry, the question of enzymology and congruence with the native cell line remained. Activity for a variety of glycosyltranferases were assayed in cell lysates of HEK293 cells and simultaneously in human oviductal cell line OE-E6/E7. The different cell lines were probed for 22 separate glycosyltranaferase activities associated with N- or O-linked glycosylation and sialyation.3 The activities in both cell lines matched the products seen in the mass spectrometry for the various modifications. Even low activities for the less abundant branched modifications were witnessed. It was concluded that both cell lines were enzymatically capable of synthesizing the multi-antennary N-glycan structures.
Taken together, the results from this study demonstrate that recombinant OVGP1 protein can be expressed in HEK293 cells. The expressed protein is heavily modified with a huge variety of glycosylations, and both HEK293 and human oviduct cell lines are capable of assembling those structures. These results open the door for future characterization of the specific roles protein glycosylation may play in the function of OVGP1 or other glycoproteins during fertilization and early embryogenesis.
1. Killian, G.J. (2004) ‘Evidence for the role of oviduct secretions in sperm function, fertilization and embryo development‘, Animal Reproduction Science, 82-83 (1),
2. Satoh, T., et al. (1995) ‘Biochemical characterization of a bovine oviduct-specific sialo-glycoprotein that sustains sperm viability in vitro‘, Biochimica et Biophysica Acta (BBA) — Molecular Cell Research, 1266 (2) (pp. 117-123)
3. Yang, X., et al. (2012) ‘Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells‘, Carbohydrate Research, 358 (1), (pp. 47-55)