Murine B cell differentiation is accompanied by programmed expression of multiple novel beta-galactoside alpha2,6-sialyltransferase mRNA forms

Abstract

ST6Gal I (β-galactoside α2,6-sialyltransferase, ST6N) elaborates the ubiquitously expressed α2,6-sialyl linkage. A number of ST6Gal I mRNA isoforms, differing only in their 5′-UT regions, is transcribed from a single mouse gene, Siat1. In B-lineage cells, α2,6-sialic acid serves as extracellular ligand for CD22, a participant in cell activation via an intracellular signaling network of tyrosine kinases and SHP phosphastase. Activation and terminal differentiation of mature B cells into plasma cells is accompanied by the appearance of at least four distinct ST6Gal I mRNA isoforms. Resting splenic B-lymphocytes isolated from 8-12 wk C56Bl/6 mice expressed almost exclusively the Exons Q+O-containing form, which is the likely homolog to the previously documented human Y+Z and rat -1+0 forms. In vitro activation using recombinant CD40-ligand and conditioned media from T-helper cells resulted in a 2-to 3-fold elevation of overall ST6Gal I mRNA abundance by Day 3. This coincided with repression of the Q+O form, and appearance of three new isoforms containing 5′-untranslated sequences X 1 , X 2 , or X 3 . The X 1 form persisted through Day 10, when the transition of B cells to plasma cells was completed as evidence by disappearance of CD22 mRNA. In contrast, the X 2 form only transiently appeared at Day 3 and declined to barely detectable levels by Day 7. Expression of the X 3 form, a minor mRNA form, paralleled the X 2 form. The divergent 5′-UT exons are dispersed over 69 kb of linear genomic space of Siat1. Mutually exclusive utilization of these 5′-UT exons in transcripts predicts separate and distinct promoter regulatory regions for each mRNA isoform.

Activation of Murine CD4+ and CD8+ T Lymphocytes Leads to Dramatic Remodeling ofN-Linked Glycans

“…Although only modest reduction of SNA binding was observed comparing native and activated CD4 cells in this study, this may be due to the fact that the residual sialylated glycans at the end of 72 h are sufficient to support significant lectin binding to other glycan classes (e.g., glycolipids). In murine B cells, increased expression of the ST6Gal I gene has been observed, mediated by a B cell-specific promoter (53). Although peak expression occurs 10 days after activation, significant increases in expression were observed within 72 h as observed in this report.…”

Role for Hepatic and Circulatory ST6Gal-1 Sialyltransferase in Regulating Myelopoiesis

“…Preliminary data indicate that the presence of extrinsic ST6Gal-1 results in ␣2,6-sialyl structures of hematopoietic cells both in vivo and in ex vivo situations. 4 Further, the hypothesis predicts that the presence of extrinsic ST6Gal-1 will have a suppressive effect in myelopoiesis, and this prediction has been verified in vitro in the present work demonstrating the inhibition of in vitro colony forming activity by addition of roughly physiological levels of ST6Gal-1 to the medium. In vivo recapitulation of this process as well as elucidation of the mechanism of ST6Gal-1 action in hematopoiesis are currently in progress and will be the subject of a separate report.…”

“…Mutant mice unable to express ST6Gal-1 are essentially devoid of ␣2,6-sialyl modifications, as evidenced by the lack of binding to the Sambucus nigra lectin (SNA) 2 that specifically recognizes these structures (1). Hepatic expression of ST6Gal-1 is principally driven by P1 (2,3), one of six independently operative promoter regions regulating transcription of the ST6Gal-1 gene (4). Another promoter, P3, is responsible for low-level ST6Gal-1 expression in the liver (5).…”

Germinal Center Marker GL7 Probes Activation-Dependent Repression of N-Glycolylneuraminic Acid, a Sialic Acid Species Involved in the Negative Modulation of B-Cell Activation

“…This result indicates that ST6GAL1 expression could be responsible for the biosynthesis of the GL7 epitope in these human B-cell lines. ST6GAL1 transfers Sia onto a Gal residue of terminal N-acetyllactosamine (LacNAc; Gal ␤1-4GlcNAc) with an ␣2,6 linkage (42), and B cells have been shown to express this enzyme (20,64). This indicates that the terminal transferase reaction by ST6GAL1, but not the supply of the substrate, is the rate-limiting step in GL7 epitope biosynthesis in these cells.…”

“…It was still not clear why GL7 failed to react with mouse mature B cells, given that these cells abundantly express ␣2,6-linked sialoglycans, as St6gal1 is also expressed in these cells (20,64). The dominant difference in sialylation between mice and humans occurs in the Sia modification at the C-5 position (60).…”