Multiple proteins from ejaculated and epididymal porcine spermatozoa bind glycan motifs found in the oviduct

Abstract

SUMMARYPreviously, we demonstrated that porcine spermatozoa bind selectively to Lewis x (Le X ) and bi-antennary a2-6 sialylated N-acetyllactosamine (bi-SiaLN) glycan motifs found in the oviduct. Recognition of these motifs mediates sperm binding to oviduct epithelial cells to form a reservoir. Here, we characterize proteins that bind to Le X and bi-SiaLN in ejaculated and cauda epididymal spermato-) and bi-SiaLN were used to locate the binding proteins on spermatozoa. Binding to N-acetyllactosamine (LN), a glycan that spermatozoa bind less, was assessed as a control. Over 60% of ejaculated and epididymal spermatozoa bound to suLe X and bi-SiaLN, but only 25% bound to LN. Specific binding to suLe X and bi-SiaLN required divalent cations. Fluoresceinated suLe X bound mostly to the plasma membrane at the apex of the head, whereas bi-SiaLN bound to a broader area of the head. Further characterization of receptors was accomplished by probing, with specific glycans, sperm proteins that had been separated by SDS-PAGE and transferred to blots. Glycan blot assays identified several suLe X -binding bands that migrated as 12-250 kDa. Migration of some of the bands that bound bi-SiaLN was similar to those that bound suLe X . A 12-kDa band protein was detected in seminal fluid and ejaculated spermatozoa, but absent in epididymal spermatozoa. In summary, we determined that: (i) multiple glycan-binding proteins recognize suLe X and bi-SiaLN motifs, (ii) specific binding required divalent cations and (iii) glycanbinding proteins were identified before and after sperm contact with accessory gland fluids. While the identity of these molecules is still unknown, this information predicts characteristics of the authentic oviduct suLe X and bi-SiaLN receptors on spermatozoa.

Lactadherin is a candidate oviduct Lewis X trisaccharide receptor on porcine spermatozoa

“…Data presented here support the hypothesis that multiple proteins coordinate binding to suLe X and potentially mediate sperm adhesion to the oviduct. This is in agreement with previous data from our group (Silva et al ., 2014) that has shown that multiple glycan-binding proteins recognize suLe X and bi-SiaLN motifs and some of these glycan-binding proteins are present before sperm contact with accessory gland fluids. While multiple proteins appear to bind suLe X , in the present work we have purified and identified ADAM5 and lactadherin as suLe X -binding proteins.…”

Regulation of Sperm Function by Oviduct Fluid and the Epithelium: Insight into the Role of Glycans

“…To characterize proteins on the plasma membrane of boar sperm that recognize Le X and bi-SiaLN motifs, sperm membrane proteins were separated by SDS-PAGE, transferred to nitrocellulose and incubated with biotinylated glycans, a 'glycan blot'. This analysis showed that sperm contained a very distinct group of proteins that recognized Le X migrating with molecular weights ranging between 12 and 250 kDa (Silva et al 2014). Binding of Le X was reduced when the Ca 2+ chelator EGTA was added, indicating that binding to at least some putative glycan receptors was Ca 2+dependent.…”

“…The 12–15 kDa proteins were absent from this preparation, and four bands from 30 to 44 kDa and two bands at 37 and 44 kDa were present that bound Le X and bi‐SiaLN, respectively (Silva et al. ). Based on similar migration during SDS‐PAGE, it appears that some proteins may recognize both glycans.…”

“…This analysis showed that sperm contained a very distinct group of proteins that recognized Le X migrating with molecular weights ranging between 12 and 250 kDa (Silva et al. ). Binding of Le X was reduced when the Ca 2+ chelator EGTA was added, indicating that binding to at least some putative glycan receptors was Ca 2+ ‐dependent.…”

Synthesis of Lewis X - O -Core-1 threonine: A building block for O -linked Lewis X glycopeptides

“…3,4,10–13 Glycoproteins expressing Le X have been identified as early mediators of the immune response against invading pathogens, 6,14,15 and are pivotal in immunomodulation, 6,16–19 regulation of the growth and angiogenesis of tumor cells, 1,4 embryogenesis of the central nervous system 3,10,11 and promotion of the formation of a spermatozoa reservoir and subsequent release from the oviduct. 20–22 DC-SIGN (dendritic cell specific ICAM grabbing non-integrin) is a counter-receptor to many Le X bearing glycoproteins, including CEACAM-1, Mac-1 and ICAM3, as well as unidentified Le X glycoproteins on invading pathogens and neutrophils. 2,7,9,23–27 In turn, the interaction of dendritic cells and neutrophils enables antigen-presenting cells to stimulate T cells and modulate adaptive immunity.…”