GlycoTCFM: Glycoproteomics Based on Two Complementary Fragmentation Methods Reveals Distinctive O‑Glycosylation in Human Sperm and Seminal Plasma

Human semen, consisting of spermatozoa (sperm) and seminal
plasma,
represents a special clinical sample type in human body fluid. Protein
glycosylation in sperm and seminal plasma plays key roles in spermatogenesis,
maturation, capacitation, sperm–egg recognition, motility of
sperm, and fertilization. In this study, we profiled the most comprehensive
O-glycoproteome map of human sperm and seminal plasma using our recently
presented Glycoproteomics based on Two Complementary Fragmentation
Methods (GlycoTCFM). We showed that sperm and seminal plasma contain
many novel and distinctive O-glycoproteins, which are mostly located
in the extracellular region (seminal plasma) and sperm membrane, enriched
in the biological processes of cell adhesion and angiogenesis, and
mainly involved in multiple biological functions including extracellular
matrix structural constituents and binding. Based on GlycoTCFM, we
created a comprehensive human sperm and seminal plasma O-glycoprotein
database that contains 371 intact O-glycopeptides and 202 O-glycosites
from 68 O-glycoproteins. Interestingly, 105 manually confirmed O-glycosites
from 25 O-glycoproteins were reported for the first time, and they
were mainly modified by core 1 O-glycans. We also found that three
highly abundant, highly complex, and highly O-glycosylated proteins
(semenogelin-1, semenogelin-2, and equatorin) may play important roles
in sperm or seminal plasma composition and function. These data deepen
our knowledge about O-glycosylation in sperm and seminal plasma and
lay the foundation for the functional study of O-glycoproteins in
male infertility.