Different glycoforms of the human GPI-anchored antigen CD52 associate differently with lipid microdomains in leukocytes and sperm membranes

Abstract

CD52 is a human GPI-anchored antigen, expressed exclusively in the immune system and part of the reproductive system (epididymal cells). Sperm cells acquire the antigen from the epididymal secretions when transiting in the epididymal corpus and cauda. The peptide backbone of CD52, consisting of only 12 aminoacids, is generally considered no more than a scaffold for post-translational modifications, such as GPI-anchor and especially N-glycosylation which occur at the third asparagine. The latter modification is highly heterogeneous, especially in the reproductive system, giving rise to many different glycoforms, some of which are tissue specific. A peculiar O-glycan-containing glycoform is also found in reproductive and immune systems. We determined to locate CD52 in microdomains of leukocytes and sperm membranes using two antibodies: (1) CAMPATH-1G, the epitope of which includes the last three aminoacids and part of the GPI-anchor of glycoforms present in leukocytes and sperm cells; (2) anti-gp20, the epitope of which belongs to the unique O-glycan-bearing glycoform also present in both cell types. Using a Brij 98 solubilization protocol and sucrose gradient partition we demonstrated that the CD52 glycoforms recognized by both antibodies are markers of typical raft microdomains in leukocytes, whereas in capacitated sperm the O-glycoform is included in GM3-rich microdomains different from the cholesterol and GM1-rich lipid rafts with which CAMPATH antigen is stably associated. The importance of the association between GM3 and O-glycans for formation of specialized microdomains was confirmed by heterologous CD52 insertion experiments. When prostasomes from human seminal fluid were incubated with rat sperm from different epididymal regions, the CD52 glycoform recognized by anti-gp20 decorated rat epididymal corpus and cauda sperm, associated with the same low-cholesterol GM3-rich sperm membrane fractions as in human sperm. The glycoforms recognized by CAMPATH-1G were not found in rat sperm. The relationship between this differential insertion and differences in glycosylation of rat and human CD52 is discussed.