(74e) The α2,3 Sialyltransferase ST3Gal-IV Regulates Human Leukocyte Binding to All Three Selectins: Distinction Between Mice and Men

Introduction: Leukocyte adhesion to the inflamed vascular
endothelium is initiated by the capture and rolling of cells on E- and
P-selectin expressed on the endothelial cells. Here, sialo-fucosylated
glycans, such as sialyl
Lewis-X (sLe^X), expressed on leukocytes act
as the selectin ligand. Such glycans are primarily
synthesized in the Golgi by the action of glycosyltransferases,
a family of enzymes that constitute ~1% of the human genome. The current paper
applies RNAi strategy to determine the human α2,3 sialyltransferases responsible
for the synthesis of P-, L- and E-selectin ligands on leukocytes. The data
reveal that, in humans, ST3Gal-IV is the dominant enzyme responsible for the
synthesis of selectin-ligands against all members of the selectin family.

Materials
and Methods: We studied all three
leukocyte-specific, human α2,3 sialyltransferases, ST3Gal-III, -IV and -VI, that attach sialic acid residues to the N-Acetyl Lactosamine  or LacNAc substrate
(Galβ1,4GlcNAc) (Figure 1). ShRNA that suppress these enzyme expression by >85% were
determined. Methods to introduce these constructs into hard-to-transfect
leukocytes (HL-60 promyelocytic cells) were developed
using lentivirus. RT-PCR confirmed gene silencing in
leukocytes. A variety of fluorescent reporters and drug selection markers were
introduced to ensure stable gene knock-down in these cells. Stable single
(ST3Gal-III¯ HL-60, ST3Gal-IV¯ HL-60, ST3Gal-VI¯ HL-60) and dual
(ST3Gal-III¯-IV¯ HL-60, and ST3Gal-IV¯-VI¯ HL-60) HL-60 knockdown cells were
created. Glycan structure analysis on these cells was performed using antibody
staining and flow cytometry. Microfluidic cell
adhesion assays measured selectin dependent cell rolling function. Results were
compared to primary neutrophils isolated from ST3Gal-IV^-/- knock-out
mice.

Results and Discussion: The ST3Gal knock-down cell lines displayed distinct
cell surface glycans and rolling pheonotypes.
Silencing ST3Gal-IV substantially reduced cell surface
CLA/HECA-452 (cutaneous lymphocyte antigen) and sLe^X
expression. ST3Gal-III has a modest role in reducing the CLA epitope
while ST3Gal-VI upregulated this epitope. In this
regard, while the CLA epitope is primarily found on leukocyte PSGL-1
(P-selectin glycoprotein ligand-1), sLe^X
is more ubiquitously expressed on other molecular entities also. Thus, all
enzymes ST3Gal-III, -IV and -VI appear to regulate the glycan structure on
PSGL-1 and also other molecular entities. When these cells were perfused over either IL-1β stimulated HUVEC monolayers or cells bearing
E-selectin (selectin associated with inflammation) or P-selectin (involved in
platelet-neutrophil interactions) or L-selectin (involved in lymphocyte homing
and leukocyte-leukocyte interaction), different enzymes were observed to contribute
to different selectin-ligand interactions. E-selectin mediated binding was
exclusively dependent on ST3Gal-IV. Silencing this gene abolished leukocyte
rolling and adhesion on stimulated HUVEC and E-selectin bearing substrates.
This observation is in marked contrast to ST3Gal-IV^-/- mice where
ST3Gal-IV only partially reduces leukocyte rolling. Human ST3Gal-IV also played
a dominant role in P-selectin dependent adhesion, unlike mice where this gene
had a partial effect. Human ST3Gal-IV and ST3Gal-VI in tandem regulated
leukocyte interaction with substrates bearing L-selectin.

Conclusions: Knocking down ST3Gal-IV alone is sufficient to
abolish E and P-Selectin mediated leukocyte recruitment in human systems. This
is markedly different from murine systems where granulocytes from ST3GalIV^-/-
mice only display partial reduction in selectin-ligand binding activity. Overall,
human ST3Gal-IV may be a viable target for anti-inflammatory therapy since it
alters leukocyte interaction with endothelial cells and platelets.