Abstract
Abstract
The Human Contraception Antibody (HCA), is a monoclonal antibody that agglutinates and immobilizes sperm, and is the active component in a contraceptive product undergoing clinical trials. To date, the HCA epitope has been described as an
N
-linked polylactosamine glycan on CD52, but this assignment has been inferred by colocalization with anti-CD52 and S19 antibodies in western blots. Here, we report that HCA binds semenogelin-galectin-3 complexes associated with sperm. Semenogelins are gel-forming, highly glycosylated proteins that are cleaved into smaller segments that adhere to sperm during semen liquefaction. We isolated HCA binding partners from seminal plasma and sperm, and semenogelin was identified in the binding fraction by mass spectrometry. Western blots demonstrated that HCA binds to a large smear of seminal plasma proteins with a higher apparent molecular mass than CD52. Flow cytometric analysis of deglycosylated sperm demonstrated that HCA binding requires sperm surface
N
-linked glycans. Despite this, HCA failed to bind any glycans represented on a comprehensive microarray. In addition to semenogelin, Galectin-3 was identified in the HCA purified sperm lysates, supporting a model in which HCA’s binding site contains semenogelin-derived glycopeptides tethered to sperm via galectins bound to an
N-
linked glycan. We found that
O
-linked glycans moieties on semenogelin contain LacNAc motifs that are recognized by galectins. Finally, depletion of sperm surface galectins reduced HCA-mediated agglutination, and this was rescued by addition of recombinant galectin-3/semenogelin-2 complexes. Collectively, our findings suggest that HCA targets an antigen that contains a semenogelin-galectin-3 complex anchored to the sperm surface via an
N
-linked glycan.
Summary sentence
We provide evidence that the Human Contraception Antibody binds to a semenogelin derived glycopeptide complex that attaches to the surface of sperm via a galectin mediated interaction; it remains unclear whether CD52 is part of the complex.
Abstract Figure
Graphical Abstract