Remodeling of marrow hematopoietic stem and progenitor cells by non-self ST6Gal-1 sialyltransferase

Abstract

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin-c-Kit+ and Lin-Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin-Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.

Keywords: Glycosylation; Hematopoiesis; Hematopoietic Stem and Progenitor Cells; Plasma; Serum; Sialic Acid; Sialyltransferase; st6gal1.

FIGURE 1.

St6gal1-dP1 animals have elevated hematopoietic capacity. A–C, HSPCs are more abundant in the St6gal1-dP1 marrow. For A, B, and C, bone marrow cells were collected from age- and sex-matched C57BL/6 (W; open bars; n = 8), St6gal1-dP1 (P; hatched bars; n = 6), and St6gal1-KO (K; solid bars; n = 8). The total (Tot) cells of one femur were counted (A). Cells were analyzed by flow cytometry for LK (B) and LSK (C) populations. D–E, St6gal1-dP1 recipients support increased marrow HSPCs. Cells from wild-type (B6.SJL-Ptprc; CD45.1) marrows were adoptively transferred into C57BL/6 (W; CD45.2; n = 5; open bars) or St6gal1-dP1 (P; CD45.2; n = 6; hatched bars) recipients. The recipient marrows were analyzed 75 days after transplantation for total CD45.1+ cells (D) and in LK (E) and LSK (F) fractions. p values were <0.05 (*) and 0.01 (**). Error bars represent S.D. G–I, exogenously added ST6Gal-1 attenuates LK cell proliferation ex vivo. LK cells from wild-type marrows were cultured in the presence of rmSCF, rmTPO, rmFlt3, rmIL-3, and rmG-CSF at an initial seeding of 100,000 LK cells. G shows total cell counts after 72 h without (open bars) or with the additions of recombinant rat ST6Gal-1 (rST6) (hatched bars) or recombinant rat ST6Gal-1 and CMP-Sia (solid bars). G is a representative of this experiment, which was repeated independently four times. H and I show flow cytometry analysis of the study from G cultivated without (H) or with (I) recombinant ST6Gal-1 (rST) and CMP-Sia using anti-c-Kit and CD11b antibodies to visualize LK (c-Kit+CD11−), committed progenitors (c-Kit+CD11+), and differentiated cells (CD11+). J, HSPCs from St6gal1-KO can be sialylated by leakage from dying cells. 10⁵ bone marrow cells of St6gal1-KO mice were hypotonically lysed for 10 min. After centrifugation, the supernatant was isolated and mixed with concentrated PBS to reconstitute the physiologic saline condition. The supernatant was added to 5 × 10⁴ LK cells and incubated with (solid line) or without recombinant ST6Gal-1 (dashed line) for 2 h. As a positive control, LK cells were also incubated with ST6Gal-1 and 100 μm CMP-Sia (shaded area). SNA reactivity was analyzed by flow cytometry.

FIGURE 2.

HSPCs from St6gal1-dP1 have reduced cell surface SNA reactivity. A–C, flow cytometry using SNA to visualize cell surface α2,6-sialyl structures was performed on total (A), LK (B), and LSK (C) bone marrow cells from C57BL/6 (black line), St6gal1-dP (dashed black line), and St6gal1-KO mice (dashed gray line). D, bone marrow cells from St6gal1-KO, which are inherently SNA-negative, were transplanted into irradiated wild-type (K>W), St6gal1-dP1 (K>P), and St6gal1-KO (K>K) recipients. The SNA profiles of the donor-derived nucleated cells with the recipient marrows were analyzed 1 week after transplantation.

FIGURE 3.

HSPCs from wild-type C57BL/6 mice have negligible endogenous ST6Gal-1. A–D, undetectable levels of ST6Gal-1 mRNA in WT marrow LK and LSK cells. Real time RT-PCR analysis was performed for ST6Gal-1, ST3Gal-3, ST3Gal-4, and ST3Gal-6, respectively, using RNA from WT LSK cells, LK cells, total bone marrow nucleated cells (BM), peritoneal macrophages (mac), splenic B220+ cells (B), and liver tissue homogenate (liv). E and F, absence of endogenous ST6Gal-1 enzymatic activity in WT LSK and LK cells. Sialyltransferase activities were measured by following transfer of CMP-[³H]Sia to Galβ1–4GlcNAc-O-Bn (LacNAc). The Siaα2,6 products (E) were separated from Siaα2,3 products (F) by SNA-agarose chromatography. Error bars represent S.D. G, HSPCs from wild-type mice acquire intrinsic ST6Gal-1 activity during differentiation. LK cells from wild-type mice were incubated in the presence of rmSCF, rmTPO, rmFlt3, rmIL-3, and rmG-CSF for 6 days. A fraction of cells was used to extract RNA before (LK) and every day during incubation. RT-PCR was performed using 50 ng of cDNA to analyze ST6Gal-1 expression. Expression of the ribosomal protein RPL32 (RPL) was analyzed as a control.

FIGURE 4.

Marrow hematopoietic cell surface α2,6-sialylation completely depends on recipient ST6Gal-1 status in bone marrow chimeras. Bone marrow cells from St6gal1-KO (KO) or WT donors, both of which were CD45.2, were transplanted into irradiated WT CD45.1 recipients. The WT into KO chimera was generated using CD45.1 WT cells and CD45.2 KO recipients. The KO donor into KO chimera used CD45.2 donors and recipients. Either total bone marrow (BM) nucleated cells (A and B) or the purified LK fraction (C) carrying the appropriate donor congenic marker was profiled for SNA (A and C) or PSL (B) reactivity by flow cytometry.

FIGURE 5.

LK cell surface SNA reactivity is not acquired by scavenging foreign SNA-positive material. A, LK cells from St6gal1-KO (KO) were incubated ex vivo in RPMI 1640 medium with 50% WT (C57BL/6) or KO mouse serum for 3 h (solid line and shaded gray area, respectively). WT LK cells were similarly incubated in the presence of WT serum and are shown as reference (dashed line). B and C, LK cells from St6gal1-KO (KO), which expressed CD45.2, were incubated ex vivo in the presence of 5-fold excess total bone marrow cells from CD45.1 WT (solid line) or KO (shaded area). After 3 or 7 days (B and C, respectively), the CD45.2 St6gal1-KO cells were examined for cell surface SNA reactivity by flow cytometry. WT LK cells similarly incubated with WT marrow cells are shown as reference (dashed line). The incubations were performed in the presence of rmSCF, rmTPO, rmFlt3, rmIL-3, and rmG-CSF as specified under “Experimental Procedures.”

FIGURE 6.

MS analysis for Siaα2,3Gal and Siaα2,6Gal on N-linked glycans of transplanted St6gal1-KO hematopoietic cells. St6gal1-KO bone marrow cells were adoptively transferred into lethally irradiated St6gal1-KO or wild-type recipients (K>K or K>W, respectively). Bone marrow St6gal1-KO cells, which were CD45.2, were recovered from the wild-type CD45.1 hosts and subjected to glycomics analysis for the presence of Siaα2,6Gal on N-linked glycans as described under “Experimental Procedures.” Lithium adducts of the parent MS¹ m/z 1421.7 ion, which is the biantennary structure containing two sialic acids in the N-glycolylneuraminic acid (Neu5Gc) form, was chosen for in-depth analysis (inset box) to generate sequentially the fragmentation ions on MS² m/z 861, MS³ m/z 456, and MS⁴ m/z 211 that corresponded to boxed areas of the parent glycan structure in the inset. Fragmentation of the “B” and “Y” bonds of the permethylated glycans generated the MS⁴ m/z 211 ion, representing the penultimate Gal residue. Further fragmentation generated the diagnostic MS⁵ scar ions; dashed lines denote the labile fragmentation sites. For Neu5Gcα2,3Gal, the MS⁵ m/z 109 and m/z 137 fragments (arrows) were produced by separate fragmentation events of the MS⁴ m/z 211 ion. For Neu5Gcα2,6Gal, the MS⁵ m/z 95 and m/z 123 fragments (circled) were produced by the same fragmentation event on the MS⁴ m/z 211 ion. The α2,6-NeuGc-specific ions m/z 95 and m/z 123 (circled) were present only on St6gal1-KO cells recovered from wild-type hosts (K>W) but not from those recovered from St6gal1-KO hosts (K>K).