Primary structure determination and physicochemical characterization of DSP-3, a phosphatidylcholine binding glycoprotein of donkey seminal plasma.

Major proteins of the seminal plasma in a variety of mammals such as bovine PDC-109, equine HSP-1/2, and donkey DSP-1 contain fibronectin type-II (FnII) domains and are referred to as FnII family proteins. To further our understanding on these proteins, we carried out detailed studies on DSP-3, another FnII protein of donkey seminal plasma. High-resolution mass-spectrometric studies revealed that DSP-3 contains 106 amino acid residues and is heterogeneously glycosylated with multiple acetylations on the glycans. Interestingly, high homology was observed between DSP-1 and HSP-1 (118 identical residues) than between DSP-1 and DSP-3 (72 identical residues). Circular dichroism (CD) spectroscopic and differential scanning calorimetric (DSC) studies showed that DSP-3 unfolds at ~45 °C and binding of phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases the thermal stability. Analysis of DSC data suggested that unlike PDC-109 and DSP-1, which exist as mixtures of polydisperse oligomers, DSP-3 most likely exists as a monomer. Ligand binding studies monitoring changes in protein intrinsic fluorescence indicated that DSP-3 binds lyso-phosphatidylcholine (K _a  = 1.08 × 10 ⁵  M ^-1 ) with ~80-fold higher affinity than PrC (K _a  = 1.39 × 10 ³  M ^-1 ). Binding of DSP-3 to erythrocytes leads to membrane perturbation, suggesting that its binding to sperm plasma membrane could be physiologically significant.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Musti J. Swamy reports financial support was provided by India Ministry of Science & Technology, Department of Science and Technology. Musti J. Swamy reports financial support was provided by University of Hyderabad.
(Copyright © 2023 Elsevier B.V. All rights reserved.)