A male genital tract-specific carbohydrate epitope on human CD52: implications for immunocontraception.

The identification of unique sperm surface epitopes that are not expressed or exposed in the female reproductive tract is a key element in the development of antibody-based contraceptives. Western blotting and immunohistochemistry were performed to define the tissue distribution of the S19 epitope, which has been proposed as a target for immunocontraception. S19 is an IgG1 murine monoclonal antibody (mAb) directed to an N-linked carbohydrate epitope on a 15-25 kDa glycoprotein, sperm agglutination antigen-1 (SAGA-1), containing a peptide core identical to that of the lymphocytic surface protein CD52. In this study, the S19 epitope was shown to be absent from human lymphocytes, demonstrating a distinction between this epitope and the CAMPATH epitope that is recognized by an antibody against the terminal tripeptide and GPI-anchor of CD52. Further tissue specificity analysis identified the S19 epitope in the epithelium of the human epididymis and vas deferens, as well as on both epididymal and ejaculated spermatozoa. In contrast, the S19 epitope was absent in the five human female reproductive tract and 18 other somatic tissues tested. These results support the use of the S19 epitope as a contraceptive immunogen and the suitability of the S19 mAb as an intravaginal contraceptive. To test the agglutinating activity of the S19 mAb in a formulation designed for vaginal use, S19 mAb were bound to the surface of Novasomes, a multilamellar liposome delivery vehicle. S19-Novasome formulations agglutinated human spermatozoa and were as effective as unbound S19 mAb, demonstrating the feasibility of spermistatic contraceptives targeted to the male reproductive tract specific carbohydrate epitope.

RASA, a recombinant single-chain variable fragment (scFv) antibody directed against the human sperm surface: implications for novel contraceptives.

BACKGROUND: A recombinant single-chain variable fragment (scFv) antibody was engineered to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), a sperm glycoform of CD52. METHODS AND RESULTS: cDNAs encoding the variable regions of the S19 [IgG(1)kappa] monoclonal antibody (mAb) were identified, linked, and cloned into the pCANTAB 5E vector. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells as a 29 kDa monomer and, remarkably, also formed multimers of approximately 60 and 90 kDa. RASA reacted with the endogenous SAGA-1 antigen by Western blot analysis, labelled the entire human sperm surface by indirect immunofluorescence, and aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination, as was also observed with the native S19 mAb. CONCLUSIONS: These results demonstrate that active recombinant antibodies can be produced to a tissue-specific carbohydrate epitope on the human sperm surface, thereby opening opportunities for novel contraceptive agents.

Differential nuclear localization of the cancer/testis-associated protein, SPAN-X/CTp11, in transfected cells and in 50% of human spermatozoa.

Cancer-testis antigens (CTAs) represent potential targets for cancer immunotherapy because these proteins are widely distributed in tumors but not in normal tissues, except testes. In this paper, we identify homology of the CTA CTp11 with SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome). On two-dimensional Western blots of human sperm extracts, SPAN-X antibodies recognized 19 spots ranging from 20 to 23 kDa with isoelectric points from 5.0 to 5.5. Differential extraction of spermatozoa demonstrated that the SPAN-X protein is highly insoluble. Only 50% of ejaculated spermatozoa exhibited SPAN-X immunofluorescent staining. Dual localization of the sex chromosomes and the SPAN-X protein demonstrated that an equal number of X- and Y-bearing spermatozoa exhibited SPAN-X staining. In transfected mammalian CV1 cells, the SPAN-Xa and SPAN-Xb proteins were localized to the nucleus and cytoplasm, respectively, by indirect immunofluorescence. On immunoblots of CV1 cells, the SPAN-Xa protein migrated at 15-20 kDa, whereas the SPAN-Xb protein migrated at a higher molecular weight of 21-22 kDa. The SPAN-X protein was ultrastructurally associated with nuclear vacuoles and the redundant nuclear envelope. SPAN-X is the first protein specifically localized to these poorly characterized structures of the mammalian sperm nucleus and provides a unique biochemical marker for investigation of their function in spermatozoa as well as the role of SPAN-X/CTp11 in human tumors.

Anti-sperm antibodies from infertile patients and their cognate sperm antigens: a review. Identity between SAGA-1, the H6-3C4 antigen, and CD52.

PROBLEM: The correlation of anti-sperm antibodies (ASA) with some instances of unexplained infertility implicates a role for these antibodies in blocking fertilization. Improved diagnosis and treatment of immunologic infertility, as well as a more complete understanding of the mechanism behind this phenomenon, are dependent on the identification and characterization of relevant sperm antigens. METHOD OF STUDY: In this article, we review literature on methods employed to identify sperm antigens using anti-sperm polyclonal and monoclonal antibodies from infertile patients and vasectomized men. Particular focus is given to approaches using human and mouse monoclonal antibodies to define the SAGA-1 human sperm antigen. RESULTS: ASA present in sera and genital tract secretions from infertile patients and vasectomized men have been employed in a variety of methods to identify sperm antigens. In an alternate approach, a monoclonal antibody (mAb), H6-3C4, was immortalized from the lymphocytes of an infertile woman who exhibited sperm-immobilizing titers. Subsequently, the sperm-agglutinating, murine S19 mAb was shown to react with the H6-3C4 cognate antigen. The H6-3C4 S19 cognate antigen, designated Sperm Agglutination Antigen-1 (SAGA-1), was characterized as a polymorphic, highly acidic, GPI-anchored glycoprotein on the surface of human spermatozoa. Purification with the S19 mAb followed by microsequencing demonstrated that the SAGA-1 core peptide is identical to CD52, a glycoprotein on the surface of human lymphocytes. Immunoblot analysis demonstrated that these two glycoproteins differed in carbohydrate composition. Thus, sperm SAGA-1 and lymphocyte CD52 represent glycoforms, glycoproteins with the same core peptide but with different carbohydrate structures. CONCLUSIONS: Autoimmunity to the SAGA-1 and/or CD52 glycoforms may lead to infertility. Structural and immunologic differences between these glycoproteins may be important factors in the etiology of immunologic infertility and other autoimmune disorders.

Evidence for a unique N-linked glycan associated with human infertility on sperm CD52: a candidate contraceptive vaccinogen.

A major objective in developing a sperm antigen-based contraceptive vaccine for humans is the discovery of sperm surface immunogens that are functionally relevant and sperm specific. The latter criterion is deemed essential to avoid the possibility of inducing autoimmune disease upon vaccination. This review presents evidence that a unique carbohydrate epitope is synthesized in the human epididymis, is attached to the core peptide of CD52, a lymphocyte differentiation marker, and is subsequently inserted into the sperm membrane via a glycosylphosphatidylinositol anchor. This unique CD52 glycoform is localized to the entire sperm surface, functions as a potent target for agglutinating and cytotoxic antibodies, and is one of the few well-defined sperm surface glycoproteins indicated in human antibody-mediated infertility.

N-linked glycan of a sperm CD52 glycoform associated with human infertility.

In a benchmark study, Isojima and colleagues established H6-3C4, the first successful heterohybridoma immortalized from the peripheral blood lymphocytes of an infertile woman who exhibited high sperm-immobilizing antibody titers. The present report demonstrates the identity between the glycoprotein antigens recognized by the human H6-3C4 monoclonal antibody (mAb) and the murine S19 mAb, generated in our laboratory to sperm agglutination antigen-1 (SAGA-1). Both mAb's recognize N-linked carbohydrate epitopes on the 15-25 kDa, polymorphic SAGA-1 glycoprotein that is localized to all domains of the human sperm surface. Treatment with phosphatidylinositol-specific phospholipase C demonstrated that SAGA-1 is anchored in the sperm plasmalemma via a GPI-lipid linkage. Immunoaffinity purification and microsequencing indicated that the core peptide of the SAGA-1 glycoprotein is identical to the sequence of CD52, a GPI-anchored lymphocyte differentiation marker implicated in signal transduction. Comparison of anti-SAGA-1 and anti-CD52 immunoreactivities revealed that the sperm form of CD52 exhibits N-linked glycan epitopes, including the epitope recognized by the infertility-associated H6-3C4 mAb, which are not detected on lymphocyte CD52. Thus, the two populations of the CD52 glycoprotein on lymphocytes and spermatozoa represent glycoforms, glycoprotein isoforms with the same core amino acid sequence but different carbohydrate structures. Furthermore, mAb's to the unique carbohydrate epitopes on sperm CD52 have multiple inhibitory effects on sperm function, including a cytotoxic effect on spermatozoa in the presence of complement. These results are the first to implicate unique carbohydrate moieties of a sperm CD52 glycoform as target epitopes in the anti-sperm immune response of an infertile woman. Furthermore, localization of CD52 on all domains of the sperm surface coupled with the multiple sperm-inhibitory effects of antibodies to its unique carbohydrate moieties suggest opportunities for immunocontraceptive development.

Effect of deletion of 5'HS3 or 5'HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice.

To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on beta-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a beta-globin locus yeast artificial chromosome, and transgenic mice were produced. Deletion of 5'HS3 resulted in a significant decrease of epsilon-globin gene expression and an increase of gamma-globin gene expression in embryonic cells. Deletion of 5'HS2 resulted in only a small decrease in expression of epsilon-, gamma-, and beta-globin mRNA at all stages of development. Neither deletion affected the temporal pattern of globin gene switching. These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites. The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.

Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of beta-globin locus YAC mice carrying human globin developmental mutants.

To test whether yeast artificial chromosomes (YACs) can be used in the investigation of mammalian development, we analyzed the phenotypes of transgenic mice carrying two types of beta-globin locus YAC developmental mutants: (i) mice carrying a G-->A transition at position -117 of the A gamma gene, which is responsible for the Greek A gamma form of hereditary persistence of fetal hemoglobin (HPFH), and (ii) beta-globin locus YAC transgenic lines carrying delta- and beta-globin gene deletions with 5' breakpoints similar to those of deletional HPFH and delta beta-thalassemia syndromes. The mice carrying the -117 A gamma G-->A mutation displayed a delayed gamma- to beta-globin gene switch and continued to express A gamma-globin chains in the adult stage of development as expected for carriers of Greek HPFH, indicating that the YAC/transgenic mouse system allows the analysis of the developmental role of cis-acting motifs. The analysis of mice carrying 3' deletions first provided evidence in support of the hypothesis that imported enhancers are responsible for the phenotypes of deletional HPFH and second indicated that autonomous silencing is the primary mechanism for turning off the gamma-globin genes in the adult. Collectively, our results suggest that transgenic mice carrying YAC mutations provide a useful model for the analysis of the control of gene expression during development.

X-ray and primary structure of horse serum albumin (Equus caballus) at 0.27-nm resolution.

The amino-acid sequence and three-dimensional structure of equine serum albumin have been determined. The amino-acid sequence was deduced from cDNA isolated from equine liver. Comparisons of the primary structure of equine serum albumin with human serum albumin and bovine serum albumin reveal 76.1% and 73.9% sequence identity, respectively. The three-dimensional structure was determined crystallographically by the molecular-replacement method using molecular coordinates from the previously determined structure of human serum albumin, to a resolution of 0.27 nm. In accordance with the primary structure, the three-dimensional structures are highly conserved. There is a root-mean-square difference between alpha-carbons of the two structures of 0.201 nm. The association constants (Ka) for the binding of 2,3,5-triiodobenzoic acid were determined by ultrafiltration methods for equine and human serum albumins to be approximately 10(4) M-1 and 10(5) M-1, respectively. Crystallographic studies of equine serum albumin reveal two binding sites for 2,3,5-triiodobenzoic acid identical with those previously reported for human serum albumin which are located within subdomains in IIA and IIIA. Details and comparisons of the binding chemistry are discussed.