The Sda/GM2-glycan is a carbohydrate marker of porcine primordial germ cells and of a subpopulation of spermatogonia in cattle, pigs, horses and llama

Klisch, Karl; Contreras, David A.; Sun, Xun; Brehm, Ralph; Bergmann, Martin; Alberio, Ramiro

doi:10.1530/rep-11-0007

Cited by 28 publications

(29 citation statements)

References 30 publications

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“…Porcine B4GALNT2 enzymatic activity has been reported from swine large intestine mucosal cells and immunostaining of the Sd a antigen has been detected in porcine primordial germ cells . To our knowledge, this is the first molecular analysis of porcine B4GALNT2 gene expression in pig tissues.…”

Section: Discussionmentioning

confidence: 82%

Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen

Byrne

Stalboerger

et al. 2014

Xenotransplantation

129

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Background Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltrasnferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. Methods The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. Results The porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes respectively. The B4GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4GALNT2 in human HEK cells (HEK-B4T) results in increased binding of antibody to the B4GALNT2 enzyme, and increased reactivity with anti-Sda and DBA. HEK-B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO:CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non-Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK-B4T cells. Conclusion The functional isolation of the porcine B4GALNT2 gene from a PAEC expression library, the pattern of B4GALNT2 gene expression and its sensitization of HEK-B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4GALNT2 produces a new immunogenic non-Gal glycan which contributes in part to the non-Gal immune response d...

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Section: Discussionmentioning

confidence: 82%

Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen

Byrne

Stalboerger

et al. 2014

Xenotransplantation

129

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“…Porcine primordial germ cells are also reported to express SDa and stain with anti-SDa antibody 90 . In contrast, cultured bovine ECs do not express B4GALNT2 ( Figure 1C), which is comparable to human and most murine strains, suggesting that clinical products produced from bovine tissues, such as heart valves, may be less likely to express B4GALNT2-dependent glycans.…”

Section: P Orcine B 4 G Alnt2mentioning

confidence: 99%

B4GALNT2 and xenotransplantation: A newly appreciated xenogeneic antigen

Byrne

Ahmad-Villiers

et al. 2018

Xenotransplantation

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Analysis of non-Gal antibody induced after pig-to-baboon cardiac xenotransplantation identified the glycan produced by porcine beta-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2) as an immunogenic xenotransplantation antigen. The porcine B4GALNT2 enzyme is homologous to the human enzyme, which synthesizes the human SDa blood group antigen. Most humans produce low levels of anti-SDa IgM which polyagglutinates red blood cells from rare individuals with high levels of SDa expression. The SDa glycan is also present on GM2 gangliosides. Clinical GM2 vaccination studies for melanoma patients suggest that a human antibody response to SDa can be induced. Expression of porcine B4GALNT2 in human HEK293 cells results in increased binding of anti-SDa antibody, and increased binding of Dolichos biflorus agglutinin (DBA) a lectin commonly used to detect SDa. In pigs, B4GALNT2 is expressed by vascular endothelial cells and endothelial cells from a wide variety of pig backgrounds stain with DBA suggesting that porcine vascular expression of B4GALNT2 is not polymorphic. Mutations in B4GALNT2 have been engineered in mice and pigs. In both species, the B4GALNT2-KO animals are apparently normal and no longer show evidence of SDa antigen expression. Pig tissues with a mutation in B4GALNT2, added to a background of alpha-1,3-galactosyltransferase deficient (GGTA1-KO) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase deficient (CMAH-KO), show reduced antibody binding, confirming the presence of B4GALNT2 dependent antibodies in both humans and nonhuman primates. Preclinical xenotransplantation using B4GALNT2 deficient donors has recently been reported. Elimination of this source of immunogenic pig antigen should minimize acute injury by preformed anti-pig antibody and eliminate an induced clinical immune response to this newly appreciated xenotransplantation antigen.

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“…In pigs, gonocytes will proliferate, migrate to the basement membrane of the tubules, and differentiate to SSCs during the first 3 months of life. These gonocytes can be identified by the marker dolichos biflorus agglutinin R150 G Manku and M Culty (DBA), highly expressed not only before PND14, but also during gonocyte differentiation between 2 and 3 weeks of age and in subsets of spermatogonia (Klisch et al 2011, Kim et al 2013. Germ cells from 2-to 3-week-old, but not younger, prepubertal pigs also expressed stagespecific embryonic antigen 1 (SSEA1; Goel et al 2007).…”

Section: Insights From Non-rodent Speciesmentioning

confidence: 99%

Mammalian gonocyte and spermatogonia differentiation: recent advances and remaining challenges

Manku¹,

Culty²

2015

Reproduction

125

105

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The production of spermatozoa relies on a pool of spermatogonial stem cells (SSCs), formed in infancy from the differentiation of their precursor cells, the gonocytes. Throughout adult life, SSCs will either self-renew or differentiate, in order to maintain a stem cell reserve while providing cells to the spermatogenic cycle. By contrast, gonocytes represent a transient and finite phase of development leading to the formation of SSCs or spermatogonia of the first spermatogenic wave. Gonocyte development involves phases of quiescence, cell proliferation, migration, and differentiation. Spermatogonia, on the other hand, remain located at the basement membrane of the seminiferous tubules throughout their successive phases of proliferation and differentiation. Apoptosis is an integral part of both developmental phases, allowing for the removal of defective cells and the maintenance of proper germ-Sertoli cell ratios. While gonocytes and spermatogonia mitosis are regulated by distinct factors, they both undergo differentiation in response to retinoic acid. In contrast to postpubertal spermatogenesis, the early steps of germ cell development have only recently attracted attention, unveiling genes and pathways regulating SSC self-renewal and proliferation. Yet, less is known on the mechanisms regulating differentiation. The processes leading from gonocytes to spermatogonia have been seldom investigated. While the formation of abnormal gonocytes or SSCs could lead to infertility, defective gonocyte differentiation might be at the origin of testicular germ cell tumors. Thus, it is important to better understand the molecular mechanisms regulating these processes. This review summarizes and compares the present knowledge on the mechanisms regulating mammalian gonocyte and spermatogonial differentiation.

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The Sda/GM2-glycan is a carbohydrate marker of porcine primordial germ cells and of a subpopulation of spermatogonia in cattle, pigs, horses and llama

Cited by 28 publications

References 30 publications

Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen

Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen

B4GALNT2 and xenotransplantation: A newly appreciated xenogeneic antigen

Mammalian gonocyte and spermatogonia differentiation: recent advances and remaining challenges

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