(52c) Silencing α(1,3)Fucosyltransferases in Human Leukocytes Reveals Differences in E-Selectin Ligand Synthesis Between Humans and Mice

Introduction: Glyosyltransferases
are a family of about 200 enzymes that constitute ~1% of the human genome. By
catalyzing the attachment of complex carbohydrate chains on protein scaffolds,
members of this enzyme family post-translationally modify a majority of mammalian
proteins. The binding of leukocyte borne, glycosylated selectin-ligands to E-,
P- and L-selectin expressed on vascular cells plays a critical role in
regulating inflammation. The α1,3 fucosyltransferase (FUT) family of
enzymes has three members called FUT4, 7, and 9 that potentially contribute to
the formation of sLe^X type glycans on
various selectin-ligands, including P-selectin glycoprotein ligand-1 (PSGL-1)
in humans. While much of the data regarding FUT function in selectin binding
has been found through use of transgenic mice knockouts, specifically the Fut4^-/-Fut7^-/-
dual knockout which completely removes binding to all selectins, important
species specific exist in selectin function in humans and mice, particularly in
E-selectin.

Materials and Methods: For the over-expression studies,
full length FUT fusion proteins with C-terminal EGFP tag were expressed in the
mammalian Golgi of HEK293T cells. Their function was
verified
using enzyme activity assays and immunocytochemistry. The PSGL-1 scaffold was
co-expressed in these cells and its effect on leukocyte adhesion function was
determined using microfluidic flow cells with substrates bearing L-/E-/P-selectin.

For the RNAi assays, shRNA sequences that reduce by >85% each of
the α1,3 FUT activities were determined. This was
done by transducing FUT specific shRNA and monitoring reduction in EGFP signal associated
with the corresponding FUT-EGFP cell lines. Results were validated using
quantitative RT-PCR. Once identified, shRNA were co-expressed with either DsRed
or Cerulean and packaged into lentivirus. Suitable promoters were then
determined to drive high levels of gene expression and RNA interference in
HL-60 cells, a human leukocytic cell line. Stable HL-60 cells lacking either one,
two or all three of the FUTs were then constructed. The ability of these FUT
knockdowns to modify selectin-dependent cell adhesion function was assayed
using flow cytometry and microfluidic assays and compared directly in our
system to neutrophils from Fut4^-/-Fut7^-/- mice.

Results and Discussion: α1,3
FUT over-expression studies show that FUT7 confer selectin binding for all
three selectins (E/P/L). FUT9 alone also enabled E-selectin binding suggesting
a previously unnoted importance for this enzyme. Knocking-down single enzymes
in HL-60 caused specific alterations to the cell surface glycan structures with
FUT4¯ HL-60 reducing cell-surface Le^X(CD15) expression, FUT7¯ HL-60 reducing sLe^X(CD15s) and FUT9¯ HL-60 up-regulating VIM-2 (CD65s) antigen expression by
2-3 fold.

Microfluidic
based rolling studies compared the rolling and attachment to E-/P-/L-selectin
for bothe the HL-60 FUT¯ cells and Fut4^-/-Fut7^-/-
mouse neutrophils run in parallel. In complete agreement with the Fut4^-/-Fut7^-/-
neutrophils, FUT7 and FUT4 co-mediated attachment, rolling and adhesion on
cell lines bearing P- or L-selectin in HL-60. This process was fully dependent
on the KPL-1 binding epitope of PSGL-1. E-selectin was the most promiscuous selectin
ligand with cell rolling studies on L-E Cells and IL-1β stimulated HUVECs demonstrating
a role for all three FUTs (FUT9>FUT7> FUT4). KPL-1 blocking or PSGL-1¯
HL-60 only reduced rolling by 40%, suggesting at least one other E-selectin
ligand on these cells was present. FUT9 played major role in E-selectin mediated
adhesion as the FUT9¯ HL-60 reduced the number of rolling and adherent cells on
E-selectin by >50% alone, >75 percent in tandem with FUT7, and >90% in
the FUT4¯7¯9¯ HL-60. The role for FUT9 in human E-selectin binding highlights
stark species-specific differences as rolling and adhesion was decreased
>95% on E-selectin in the Fut4^-/-Fut7^-/- mouse neutrophils.

Conclusions: This study introduces novel molecular
engineering tools to study both gain and loss of glycosyltransferase function
in leukocytes and related cell adhesion function. To this end, data generated
using these methods highlight a critical and predominant role for FUT9, in
addition to FUT4 and FUT7, in the synthesis of ligands for E-selectin in human
leukocytes. This is in contrast to the observation that all selectin binding
function (including E-selectin) is lost in Fut4^-/-Fut7^-/-
mouse neutrophils. Thus, we observe notable species-specific differences in FUT
and E-selectin ligand function that may be considered during the design of
anti-inflammatory therapies that target the selectins, PSGL-1, or
fucosyltransferases.