Abstract
Sperm motility and maturation are known to be affected by a host of factors encountered en route in both male and female genital tracts prior to fertilization. Using a concerted proteomics and glycomics approach with advanced mass spectrometry-based glycan sequencing capability, we show in this work that 24p3, an abundant mouse uterine luminal fluid (ULF) glycoprotein also called lipocalin 2 (Lcn2), is highly fucosylated in the context of carrying multiple Lewis X and Y epitopes on complex type N-glycans at its single glycosylation site. The predominance of Lewis X/Y along with Neu5Acalpha2-6 sialylation was found to be a salient feature of the ULF glycome, and several other protein carriers were additionally identified including the highly abundant lactotransferrin, which is N-glycosylated at two sites, both with a similar range of highly fucosylated N-glycans. A comparative glycomics analysis of the male genital tract fluids revealed that there is a gradient of glycomic complexity from the cauda to caput regions of the epididymis, varying from high mannose to sialylated complex type N-glycans but mostly devoid of fucosylation. The seminal vesicle fluid glycome, on the other hand, carries equally abundant multimeric Lewis X structures but is distinctively lacking in additional fucosylation of the terminal galactose to give the Lewis Y epitope typifying the glycome of female ULF. One-dimensional shotgun proteomics analysis identified over 40 proteins in the latter, many of which are reported for the first time, and a majority are notably involved in immune defense and antigen processing. Further sperm binding and motility assays suggest that the Lewis X/Y epitopes do contribute to the sperm motility-enhancing activity of 24p3, whereas lactotransferrin is largely inactive in this context despite being similarly glycosylated. These findings underline the importance of glycoproteomics in delineating both the specific glycan structures and their carriers in assigning glycobiological functions.
Highlights
Sperm motility and maturation are known to be affected by a host of factors encountered en route in both male and female genital tracts prior to fertilization
By adopting a concerted proteomics and glycomics analyses, we report here the identification of several secretory components not known previously to be associated with the uterine luminal fluid and show that Lewis X (Lex) and Lewis Y (Ley) are the major fucosylated epitopes of its constitutive glycoproteins including 24p3 and lactotransferrin
Preparation of 24p3, Uterine Luminal Fluid (ULF), Seminal Vesicle Fluid (SVF), Epididymal Fluid (EF), and Spermatozoa—uterine luminal fluid (ULF) was collected from 3-week-old female mice pretreated with diethylstilbestrol, and 24p3 glycoprotein was purified thereof as described previously [16]
Summary
Preparation of 24p3, Uterine Luminal Fluid (ULF), Seminal Vesicle Fluid (SVF), Epididymal Fluid (EF), and Spermatozoa—ULF was collected from 3-week-old female mice pretreated with diethylstilbestrol, and 24p3 glycoprotein was purified thereof as described previously [16]. Lectin Enrichment and Shotgun Proteomics Identification of De-Nglycosylated Peptides—Alternatively the tryptic peptides from a total of 150 g of ULF proteins were desalted using a C18 Sep-Pak cartridge and subjected to lectin enrichment using the A. aurantia lectinagarose beads (Vector Laboratories) equilibrated in buffer containing 10 mM Tris-Cl, pH 7.5, and 150 mM NaCl. Peptide sample dissolved in the equilibration buffer was incubated overnight at 4 °C with the lectin beads. After further overnight N-glycosidase F treatment in 50 mM ammonium bicarbonate, pH 8.5, at 37 °C, peptide samples were again desalted and separated from the released N-glycans by C18 Sep-Pak cartridge (Waters) and subjected to shotgun proteomics identification by nano-LC-nano-ESI-MS/MS analysis on the Micromass Q-TOF Ultima API mass spectrometer as described above for gel-based analysis but with the added criterion of Asn to Asp conversion as one of the variable modifications for database searching.
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