Purification and structural analysis of sperm CD52, a GPI-anchored membrane protein.

Abstract

One of the most prominent changes during epididymal passage is the appearance on human spermatozoa of the so-called “major maturation-associated” antigen HE5 or sperm CD52 (Hale et al., 1993; Kirchhoff, 1996). The mRNA is expressed only post-testicularly by the epididymal epithelial cells. The cDNA sequence turned out to be colinear with that of the lymphocyte surface antigen CD52 (Xia et al., 1991), suggesting that it encodes a glycosylphosphatidylinositol (GPI)-anchored membrane protein with an unusually small peptide core, consisting of only 12 amino acids, which contains a large N-linked carbohydrate moiety. Because the peptide core seemed to be so small and poorly conserved among mammals, we assumed that the function of this molecule may reside in this N-glycan. The structure of the carbohydrate moiety of the corresponding lymphocyte antigen has been studied previously: it consists of a large, partly sialylated tetraantennary polylactosamine core (Treumann et al., 1995). We extracted the antigen from human sperm, seminal plasma, epididymal tissue, and cauda epididymal fluid. It was recognized by the monoclonal antibody CAMPATH- 1, raised against the lymphocyte antigen. The epitope consists of the GPI-anchor plus the three last C-terminal amino acids of the peptide core. Partially purified sperm CD52 migrated as multiple bands during SDS-electrophoresis, differences in electrophoretic mobility possibly due to differences in glycosylation and/or association with other proteins/glycolipids. To analyse its glycosylation pattern, sperm CD52 was digested with N-glycanase F in a time-course experiment, and detected by Western blotting employing the CAMPATH- 1 antibody. Prior to digestion, the antigen seemed to consist of several glycoforms as visualized by five bands of different electrophoretic mobility (microheterogeneity between 15 and 23 kDa). In the course of the deglycosylation reaction, the microheterogeneity disappeared, and a single intensely stained band appeared at approximately 6 kDa, showing that sperm CD52 is N-glycosylated, and that the deglycosylated form was recognized even better by the antibody. The structure of the N-glycan was further studied employing two biotinylated lectins, MAA (from Maackia amurensis) and SNA (from Sambucus nigra), showing a specific affinity for terminal sialic acids in α-2,3- or 2,6-position. SNA detected a pattern of multiple protein bands congruent to that recognized by CAMPATH-l. The MAA detection pattern was different in that it was deplete of the fastest migrating forms. From this pattern it was concluded that all sperm CD52 glycoforms detected on Western blots contained terminal 2,6-linked sialic acid. Additionally, the slower migrating forms contained 2,3-linked sialic acid. To study possible changes in glycosylation during capacitation, Percoll gradient-purified, in vitro capacitated human sperm were compared with a non-capacitated control, employing antibody and lectin binding. Comparing equal sperm counts, CD52 was shown to persist on the sperm surface during 6h of in vitro capacitation, and was still present after a 24h incubation. Moreover, the glycosylation pattern as detected by the two different lectins did not change.KeywordsSialic AcidHuman SpermCarbohydrate MoietyGlycosylation PatternLymphocyte AntigenThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.