Role for hepatic and circulatory ST6Gal-1 sialyltransferase in regulating myelopoiesis

Abstract

Recent findings have established a role for the ST6Gal-1 sialyltransferase in modulating inflammatory cell production during Th1 and Th2 responses. ST6Gal-1 synthesizes the Sia(alpha2,6) to Gal(beta1,4)GlcNAc linkage on glycoproteins on cell surfaces and in systemic circulation. Engagement of P1, one of six promoter/regulatory regions driving murine ST6Gal-1 gene expression, generates the ST6Gal-1 for myelopoietic regulation. P1 utilization, however, is restricted to the liver and silent in hematopoietic cells. We considered the possibility that myelopoiesis is responsive to the sialylation of liver-derived circulatory glycoproteins, such that reduced alpha2,6-sialylation results in elevated myelopoiesis. However, 2-dimensional differential in gel electrophoresis (2D-DIGE) analysis disclosed only minimal alterations in the sialylation of sera glycoproteins of ST6Gal-1-deficient mice when compared with wild-type controls, either at baseline or during an acute phase response when the demand for sialylation is greatest. Furthermore, sera from ST6Gal-1-deficient animals did not enhance myelopoietic activity in ex vivo colony formation assays. Whereas there was only minimal consequence to the alpha2,6-sialylation of circulatory glycoproteins, ablation of the P1 promoter did result in strikingly depressed levels of ST6Gal-1 released into systemic circulation. Therefore, we considered the alternative possibility that myelopoiesis may be regulated not by the hepatic sialyl glycoproteins, but by the ST6Gal-1 that was released directly into circulation. Supporting this, ex vivo colony formation was notably attenuated upon introduction of physiologic levels of ST6Gal-1 into the culture medium. Our data support the idea that circulatory ST6Gal-1, mostly of hepatic origin, limits myelopoiesis by a mechanism independent of hepatic sialylation of serum glycoproteins.

FIGURE 1.

Serum glycoproteins of ST6Gal-1-deficient animals are not undersialylated. Serum pooled from 5 animals from each of the three genotypes, WT, Siat1ΔP1 (ΔP1), or Siat1-null (KO), were collected, albumin-depleted, and separately labeled with CyDyes Cy-3, Cy-5, and Cy-2, respectively, before combining together for two-dimensional polyacrylamide gel electrophoresis as detailed in “Materials and Methods.” The analysis was performed on sera collected from animals at rest (0 h) or during an APR 72 h after subcutaneous injection of turpentine (72 h). Shown are the 2D-PAGE separation signal for each of the three dye channels, with the molecular weight and isoelectric directions represented as vertical and horizontal dimensions, respectively.

FIGURE 2.

SiatΔP1 serum glycoproteins are positive for α2,6-sialic acids. A, protein polyacrylamide gel blot of WT, Siat1ΔP1 (ΔP1) and Siat1-null (KO) serum proteins (0.05 μl from a pooled serum of 5 animals) at rest (0), at 48 h (48), or at 72 h (72) after turpentine elicited APR were separated on SDS-polyacrylamide gels were probed with SNA to visualize α2,6-linked sialic acid-containing glycoproteins. Quantification of (A) total SNA signal and (B) total MAA signal from Western blots, with relative signals reference to the wild-type signal of 1. Sera from 5 individual animals were used for each determination utilizing ImageQuant software, where (*) denotes p < 0.05 based on Student's t test.

FIGURE 3.

2D-DIGE comparative analysis of α2,6-sialic acid-containing wild-type and Siat1ΔP1 serum glycoproteins. A, sera pools from 5 animals of WT and from Siat1ΔP1 (ΔP1) mice at rest were de-albuminized and minimally labeled with CyDyes (wild-type, green; Siat1ΔP1, red; overlap, yellow). The combined labeled wild-type and Siat1ΔP1 pools were subjected to SNA-agarose purification for the α2,6-sialic acid containing glycoprotein fraction, and subjected to 2D-DIGE on a single 2D gel. B, differential quantitation of spots was performed utilizing DeCyder software to illuminate protein entities with genotype-dependent shift in isoelectric distribution (horizontal dimension). Shifts in the isoelectric distribution in the α2,6-sialyl glycoproteins imply differences in the overall levels of sialic acid modifications, where each sialic acid moiety confers an additional negative charge. Some α2,6-sialic acid-containing glycoproteins with reduced overall sialylation in Siat1ΔP1 animals are hemopexin (a), complement a/b (b), α2-macroglobulin (c and d), with identities determined by mass spectrometric identification as outlined in “Materials and Methods.” Shifts in the isoelectric distribution of the above mentioned glycoproteins, where each data point represents one corresponding spot on panel A, are displayed in panel B as signal strength ratios of wild-type versus Siat1ΔP1 along the isoelectric dimension.

FIGURE 4.

RT-PCR analysis of selected hepatic sialyltransferase mRNAs. Expression of (top left) total ST6Gal-1 mRNA, (top right) P1-transcribedST6Gal-1 mRNA that contains the unique Exon H sequence (2), and (bottom) selected hepatic sialyltransferases were measured by real-time PCR relative to the expression of RPL32, a ribosomal protein, as a reference standard. RT-PCR analysis was performed on liver samples from WT, Siat1ΔP1 (P1) or Siat1-null (KO) strains during either rest (T0) or 72 h after turpentine elicited APR (T72). The determinations were based on 5 animals from each of the strains for each of the time points, where (*) denotes p < 0.05 by Student's t test.

FIGURE 5.

Serum enzymatic activity of selected gal-sialyltranferases during an APR. Serum was harvested from WT, Siat1ΔP1 (P1), or Siat1ΔP1 (KO) strains at baseline (T0) or 72 h after turpentine-elicited APR (T72). Sera from 4 animals of each strain were pooled and assessed for sialyltranferase activity by CMP-[³H]NeuNAc and an artificial acceptor substrate as described in Materials and methods. Acceptor A enzymatic product was further separated by SNA-agarose to distinguish the α2,6- and α2,3-sialylated products. The chart indicates the chemical structure of each acceptor and the sialyltransferase preferences for each of these acceptor substrates.

FIGURE 6.

Attenuation of ex vivo colony formation by ST6Gal-1. Bone marrow cells were extracted from resting animals, placed into MethoCult (semi-solid media), incubated for 4–7 days. Colonies were assessed visually. Panel A, CFU of WT and Siat1ΔP1 (ΔP1) bone marrow cells, with the difference yielding a statistical significance of p < 0.05 by Student's t test. Panel B, wild-type bone marrow cell cultures were supplemented with 10% (v/v) of PBS, or plasma from WT, Siat1ΔP1 (ΔP1), or Siat1-null (KO) animals at rest. Panels C and D, wild-type bone marrow cells were cultured in the absence or presence of recombinant ST6Gal-1 (rST6G). rST6G was added to yield a final sialyltransferase activity of 11 fmol/h/μl, which was roughly equivalent to level present in plasma of wild-type animals during APR. Each point was performed in duplicate (D) or triplicate (C) as shown. Colony classification as follows: G, granulocyte; GM, granulocyte/monocyte; M, monocyte; Eos, eosinophil; Other, non-eosinophil. The rST6G supplementation study has been repeated >3 times, and a representative experiment is presented. Heat inactivated rST6G resulted in no suppression of clonogenic activity (data not shown). Cultures in panels A–C were supplemented with IL-3, and G-CSF; cultures in panel D were supplemented with IL-5.