Top–down mass spectrometry reveals new sequence variants of the major bovine seminal plasma protein PDC‐109

Abstract

The major protein of bovine seminal plasma, PDC-109, is a 109-residue polypeptide that exists as a polydisperse aggregate under native conditions. The oligomeric state of this aggregate varies with ionic strength and the presence of lipids. Binding of PDC-109 to choline phospholipids on the sperm plasma membrane results in an efflux of cholesterol and choline phospholipids, which is an important step in sperm capacitation. In this study, Fourier transform ion cyclotron resonance mass spectrometry was used to analyze PDC-109 purified from bovine seminal plasma. In addition to the previously known PDC-109 variants, four new sequence variants were identified by top-down mass spectrometry. For example, a protein variant containing point mutations P10L and G14R was identified along with another form having a 14-residue truncation in the N-terminal region. Two other minor variants could also be identified from the affinity-purified PDC-109. These results demonstrate that PDC-109 is naturally produced as a mixture of several protein forms, most of which have not been detected in previous studies. Native mass spectrometry revealed that PDC-109 is exclusively monomeric at low protein concentrations, suggesting that the protein oligomers are weakly bound and can easily be disrupted. Ligand binding to PDC-109 was also investigated, and it was observed that two molecules of O-phosphorylcholine bind to each PDC-109 monomer, consistent with previous reports.

Lectin-Binding Specificity of the Fertilization-Relevant Protein PDC-109 by Means of Surface Plasmon Resonance and Carbohydrate REcognition Domain EXcision-Mass Spectrometry

“…Evidence from the literature points out that PDC-109 is naturally produced as a mixture of several protein forms [ 28 ] and aggregation states [ 29 ] that play an important role in modulating its interaction with other biomolecules. This would allow concluding that, whichever of the situations proposed in Figure 6 applies –including a combination thereof–, PDC-109 acts as a lectin-like molecule albeit with quite unique features that, though not abolishing its ability to recognize carbohydrates, give rise to unusual SPR sensorgrams.…”

“…Specifically, the interaction with common mammal epitopes has been qualitatively and quantitatively characterized by SPR, with a larger affinity found for the Fuc-α1,4-GlcNAc disaccharide, a result in agreement with previous studies which proposed that, during sperm reservoir formation, bovine sperm binds to an oligosaccharide ligand of the oviductal epithelium similar to the Lewis a epitope (Galβ1-3[Fucα1-4]GlcNAc) [ 15 , 30 ]. On a different note, it is worth mentioning that unusual PDC-109 SPR sensorgrams posing difficulties for kinetic adjustment most likely reflected intrinsic PDC-109 properties, confirming that this protein is naturally a mixture of various isoforms and aggregation states that play an important role in modulating the interaction with other biomolecules [ 28 , 29 , 37 , 38 , 39 ]. In this context, self-aggregation of PDC-109 could be driven by the Neu5Ac-Hex-HexNAc trisaccharide found in roughly 50% of its isoforms in circulation.…”

EThcD and 213 nm UVPD for Top-Down Analysis of Bovine Seminal Plasma Proteoforms on Electrophoretic and Chromatographic Time Frames

“…6 Many seminal plasma proteins with biological relevance are smaller than 35 kDa, 1 which makes them suitable candidates for TDMS applications. TDMS has been used to investigate sequence variants of the bovine seminal plasma PDC-109, 19 but to our knowledge there have been no systematic TDMS investigations of seminal plasma.…”