1. RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
The binding of adhesion molecules of the selectin family to carbohydrate ligands
expressed on leukocytes is a critical process that regulates both immunity and inflammation.
Among the glycoprotein ligands, a single O-linked glycan attached to Thr-57 at the N-terminus
of the leukocyte surface antigen P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is
important since it binds all the selectins (L-, E-, and P-selectin) with high affinity under fluid
shear. Determination of the precise glycan(s) at this site by traditional biochemical methods
is complicated due the presence of 71 additional potential sites for O-glycosylation on this
mucinous protein.
To overcome this limitation, we developed a PSGL-1 variant ‘19Fc’ which contains the
single O-glycan at Thr-57. 19Fc was expressed in HL-60 (human leukocytic cells), HEK293T,
and HEK293T cells carrying human FUT7 enzyme. MALDI-MS/MS analysis of O-glycans released
from 19Fc revealed the O-glycan distribution at the N-terminus of PSGL-1. Additionally, MS
analysis demonstrated the presence of the sialyl Lewis-X (sLeX) glycan on a core-2 backbone
and also the disialylated T-antigen structure (Neu5Acα2,3Galβ1,3[Neu5Acα2,6]GalNAc).
Glycoproteomic analysis confirmed the site-specific expression of these O-glycans at Thr-57.
This data along with some previous literature suggest a competition between core-2 GlcNAc
transferase (C2GnT-I) and ST6GalNAc transferase enzymes may regulate the relative
expression of sLeX and disialylated T-antigen at the N-terminus of PSGL-1. To address this
hypothesis, HL-60 cells were transduced to overexpress either ST6GalNAc-1, -2, or -4. Over-
expression of ST6GalNAc-2 (ST6GalNAc2+HL60) abrogated cell surface sLeX and CLA
expression. It also reduced the number of leukocytes on P-, L-, and E-selectin bearing
substrates by 86%, 85%, and 67%, respectively, in a flow chamber based assay.
ST6GalNAc2+HL60 cells also displayed higher median rolling velocities to P-, L- and E-selectin
by 79%, 146% and 96%, respectively. ST6GalNAc-2 co-localizing with C2GnT-1 further
demonstrating the spatial location of the two enzymes enables their competition with each
other for their common substrate, Galβ1,3GalNAc.
Overall, the study provides the first detailed distribution of O-glycans at the N-
terminus of PSGL-1. The data demonstrates that a competition between C2GnT-I and
ST6GalNAc transferase regulates the synthesis of the selectin-ligand at the N-terminus of P-
selectin glycoprotein ligand-1.
Abstract
Introduction
PSGL-1 has:
• 73 potential sites for O-linked glycosylation (brown circles)
• 3 sites for N-glycosylation (purple squares)
• N-terminal residues include three sulfated tyrosines (S)
• a sialofucosylated O-glycan at Thr-57 (red box).
The constructed PSGL-1 variants include either 19- or 76- N-
terminal residues followed by the human IgG1 Fc, PSGL-1
transmembrane and cytoplasmic sections.
Results
• Quantitative analysis of O-glycan distribution at the N-
terminus of PSGL-1 was determined using the molecular
probe 19Fc and high sensitivity MALDI-MS
• Site specific expression of core-2 sLeX and disialylated T-
antigen was confirmed by glycoproteomic analysis
• HL-60 cells over-expressing ST6GalNAc-2 display:
• Reduced sLeX and CLA cell surface expression
• Reduced binding to P-selectin on activated
human platelets in a cone-and-plate viscometer
assay at shears ranging from 350/s to 4650/s
• Reduced cell rolling and higher median rolling
velocities on substrates bearing P-, L-, and E-
selectin
• 19Fc from ST6GalNAc2+HL60 displayed reduced P-
selectin binding function and low sLeX at the N-terminal
O-glycan
• ST6GalNAc-2 and C2GnT-I co-localize in Golgi
Acknowledgements
NIH, American Heart Association, Mark Diamond Research Fund, UB
Presidential Fellowship, and all the members of the Neelamegham lab for
their guidance and support.
– Protein carbohydrate binding contributes to many cell-cell interactions
– PSGL-1 binding to selectins contribute to multistep process of leukocyte
recruitment.
– Studying this interaction can give insight into many inflammatory and
thrombotic disorders including psoriasis, Crohn’s disease, rheumatoid arthritis,
asthma, arteriosclerosis and ischemia reperfusion
1From the Department of Chemical and Biological Engineering and 2The NY State Center for Excellence in Bioinformatics and Life Sciences, The State University of New York, Buffalo, NY 14260, USA
3Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College, London SW7 2AZ, UK
Chi Lo1, Aristotelis Antonopoulos3, Stuart Haslam3, Anne Dell3, Sriram Neelamegham1,2
ST6GalNAc-2 regulates leukocyte selectin-ligand synthesis on human P-selectin glycoprotein ligand-1
S
42 118 279 321 341 412
Transmembrane
domain
Cytoplasmic
domain
Decamer repeats
Human Fc
His-tag
19FcTM
76FcTM
WT PSGL-1
S
S
N-glycan
O-glycan
19Fc[HL-60]
19Fc[HEK293T]
19Fc[FUT7+HEK293T]
97.4
66.2
31
45
5’ LTR 3’ LTR
VWFspCMV R U5 19Fc U3 R U5EF1α
0
20
40
60
80
100
0 1 2 3 4
Bead-platelet
adhesion(%)
Time (min)
19Fc beads
19Fc T57A beads
19Fc beads + KPL1
Control beads
Glycoproteomic analysis in CID mode using an LTQ-Orbitrap mass spectrometer identified
core-2 sLeX and di-sialyl T-antigen at Thr-57 of PSGL-1. m/zmeas and m/zcalc are within 20 ppm.
• Lentiviral expression of ST6GalNAc along with DsRED (red fluorescent protein) reporter using an
internal ribosome entry site (IRES). Transduced cells appear red under fluorescence microscopy.
• RT-PCR analysis and enzymatic studies (not shown) confirm overexpression of these genes
• ST6GalNAc2+HL60 and ST6GalNAc4+HL60 reduced cell surface CLA and sLeX expression, as
measured using flow cytometry
Control ST6GalNAc1+ ST6GalNAc2+ ST6GalNAc4+
BrightfieldDsRED
CMV R U5
5’LTR 3’LTR
U3DsREDEF1α ST6GalNAc R U5IRES
• HL-60 and its variants were sheared with TRAP-6 activated platelets at 650/s.
• ST6GalNAc2+HL60 displayed reduced adhesion to platelets compared to wild-type HL60 cells at all
additional shear rates tested
• HL-60 and its variants were perfused over CHO-P cells, recombinant L-selectin, and E-selectin
bearing L-cells at a wall shear stress of 1 dyne/cm2
• ST6GalNAc2+HL60 reduced the number of rolling cells on P-, L-, and E-selectin by 86%, 85%, and
67%, respectively
• ST6GalNAc2+HL60 exhibited increased median rolling velocities to P-, L- and E-selectin by 79%,
146% and 96%, respectively
0%
20%
40%
60%
80%
100%
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
80
90
100
More
Rollingcell%
Rolling velocity (µm/s)
HL60
ST6GalNAc1⁺
ST6GalNAc2⁺
ST6GalNAc4⁺
1dyne/cm2
0
20
40
60
80
100
Cells/mm2
Rolling
Adherent
*
* *
HL-60 mutants
0
25
50
75
100
Cells/mm2
Rolling
Adherent
HL-60 mutants
*
*
**
0%
20%
40%
60%
80%
100%
0
10
20
30
40
50
60
70
80
90
100
110
120
140
160
More
Rollingcell%
Rolling velocity (µm/s)
HL60
ST6GalNAc1⁺
ST6GalNAc2⁺
ST6GalNAc4⁺
1dyne/cm2
0
40
80
120
Cells/mm2
Rolling
Adherent
HL-60 mutants
*
*
*
*
*
*
0%
20%
40%
60%
80%
100%
Rollingcell%
Rolling velocity (µm/s)
HL60
ST6GalNAc1⁺
ST6GalNAc2⁺
ST6GalNAc4⁺
1dyne/cm2
• 19Fc-ST6GalNAc2+ were immobilized on anti-human IgG polystyrene beads
• 19Fc-ST6GalNAc2+ bead binding to platelets was significantly reduced compared to 19Fc-beads
in the viscometer assay
• MS spectra shows marked reduction in core-2 sLeX structure on 19Fc-ST6GalNAc2+ compared to
19Fc
19Fc[ST6GalNAc2+]
* * * * *
0
20
40
60
80
100
0 50 100 150 200
Time (sec)
Control beads
19Fc beads
19Fc-ST6GalNAc2⁺ beads
19Fc beads + KPL1
19Fc-ST6GalNAc2⁺ beads + KPL1
Bead-platelet
adhesion(%)
• C2GnT-I-GFP and ST6GalNAc2-DsRED fusion proteins transduced into HEK293T
cells show co-localization is noted (yellow regions in the merged image) in
confocal microscope image
• Model suggesting competition between ST6GalNAc2 and C2GnT for the T-
antigen (Galβ1,3GalNAc) substrate.
C2GnT-I-GFP ST6GalNAc2-DsRED
Merge Merge + DIC
5’ LTR 3’ LTR
ST6GalNAc2CMV R U5 DsRED U3 R U5CMV
5’ LTR 3’ LTR
C2GnT-1CMV R U5 U3 R U5CMV GFP
Nature Rev. Immunol. 3, 569-581, 2003.
P-selectin
glycoprotein
ligand-1 (PSGL-1)
PSGL-1 in the Inflammatory Response
Characterization of 19FcTM and 76FcTM by flow cytometry
• CLA epitope is augmented upon FUT7 expression
• P-selectin IgG binding is noted upon co-expression of
FUT7 with either wild-type PSGL-1, 19FcTM or 76FcTM.
Lentiviral Expression of 19Fc in HL-60 cells
200 400 600 800 1000 1200 1400 1600 1800 2000
0
10
20
30
40
50
60
70
80
90
100
F L P E T E P P R A M M D
o o
F L P E T E P P R A M M D
o o
+2
1124.142
F L P E T E P P R A M M D
o o
+2
898.093
291.983
F L P E T E P P R A M M D
o o
+2
1042.852
F L P E T E P P R A M M D
o o
+2
978.691
F L P E T E P P R A M M D
o o
+2
795.69
F L P E T E P P R A M M D
o o
+3
750.143
F L P E T E P P R A M M D
o o
+3
599.512
F L P E T E P P R A M M D
o o
+3
696.565
F L P E T E P P R A M M D
o o
+3
652.801
454.031 657.301
366.153
y12
+3
797.784
y12
+2
1196.582
y12
+3
700.778
y12
+2
1050.656
y12
+3
604.336
y12
+2
906.039
y12
+2
969.854
y12
+2
824.402
y12
+2
723.014
L P E T E P P R A M M D
o oy12
y12
L P E T E P P R A M M D
o o
L P E T E P P R A M M D
o oy12
L P E T E P P R A M M D
o oy12
L P E T E P P R A M M D
o oy12
L P E T E P P R A M M D
o oy12
m/zmeas =847.017
m/zcalc =847.016
z= 3
%Intensity
Mass (m/z)
200 400 600 800 1000 1200 1400 1600 1800 2000
0
10
20
30
40
50
60
70
80
90
100
T E P P R A M M D
o o
+2
1124.276
T E P P R A M M D
o o
+2
868.433
366.23
803.284
860.133
T E P P R A M M D
o o
+2
905.72
T E P P R A M M D
o o
+2
978.719
T E P P R A M M D
o o
+2
1042.818
T E P P R A M M D
o o
+2
1051.172
T E P P R A M M D
o o
+2
1079.59
T E P P R A M M D
o o
+2
1197.14
T E P P R A M M D
o o
1445.468
657.314
T E P P R A M M D
o o
+2
723.126
T E P P R A M M D
o o
1736.475
T E P P R A M M D
o o
1282.703
Mass (m/z)
%Intensity
T E P P R A M M D
o o
m/zmeas = 1269.515
m/zcalc = 1269.486
z = 2
WT
HEK293T
2.5 0.4
10.4 3.5
6.4 3.6
6.7 2.1
85.9 31.5
FUT7+
19.8 2.6
12.0 3.4
6.8 4.0
6.3 2.1
85.9 70.1
19FcTM+
3.1 0.3
10.7 0.5
274.5 59.6
198.3 73.0
119.6 54.0
FUT7+
19FcTM+
17.4 5.7
15.6 0.2
263.9 58.4
261.0 66.5
334.6 96.0
76FcTM+
2.9 0.2
10.5 0.6
925.7 194.7
957.0 335.2
152.0 67.5
FUT7+
76FcTM+
20.1 3.0
24.0 2.4
614.2 110.1
895.7 67.0
530.8 201.1
23.1 7.3
28.2 9.2
6.7 3.5
177.3 93.4
305.9 15.1
Human IgG/
PSGL-1
P-selectin
binding
CLA / CHO-
131
103102101100 104103102101100 104 103102101100 104
100
75
25
50
0
100
75
25
50
0
100
75
25
50
0
100
75
25
50
0
100
75
25
50
0
100
75
25
50
0
100
75
25
50
0
CountCountCountCountCountCountCount
FUT7+
WT-
PSGL-1+
MS Reveals Microheterogeneity of 19Fc O-glycan
ST6GalNAc Transferase Over-expression in HL-60 Cells Alters
Surface Glycan Structures
PSGL1 VIM-2 LeX sLeXCLA
0
40
80
120
160
200
Meanfluorescenceintensity
HL60
ST6GalNAc1⁺
ST6GalNAc2⁺
ST6GalNAc4⁺
* *
*
Over-expression of ST6GalNAc-2 Reduces P-selectin
dependent leukocyte-platelet adhesion
19Fc Expressed from ST6GalNAc2+ Show Low sLeX
C2GnT-I
ST3Gal-I
ST6GalNAc2
α2,3
β1,6
β1,4 α1,3
α2,3 β1,3
β1,6α2,3 β1,3
β1,3
β1,6β1,3α2,3 β1,3
α2,3 β1,3 α2,6
β1,3 α2,6
…
m/z = 895.2
(major product)
m/z = 1256.4
m/z = 1879.7
m/z = 895.2
(minor product)
– Develop a tool to study site specific glycosylation
• 19Fc protein purification from HL-60, HEK293T and FUT7+HEK293T cells
• MALDI-TOF/TOF glycomics analysis to determine glycan
microhetergeneity at Thr-57
• Glycoproteomics to validate identification of site-specific
glycosylation
– Test the hypothesis that ST6GalNAc transferase over-expression reduces
the biosynthesis of functional selectin-ligands at the N-terminus of PSGL-1
• Over-expression of ST6GalNAc-1, -2, and -4 in HL-60 cells
• Cell surface glycan characterization by flow cytometry
• Assess P-selectin binding by shearing modified HL-60 cells with
stimulated platelets in a cone-and-plate viscometer
• Assess P-, L-, and E-selectin binding by perfusing cells over selectin
bearing substrates in a flow chamber assay
• Expression and purification of 19Fc from ST6GalNAc2+HL60
– Assess P-selectin binding function
– Perform MALDI-TOF/TOF glycomics analysis
• Confocal microscopy to confirm co-localization of C2GnT-I and
ST6GalNAc2
Figure from Carlow, D.A., et al., PSGL-1 function in immunity and steady
state homeostasis. Immunol Rev, 2009. 230(1): p. 75-96.
EF1α promoter and VWF signal peptide were
used to drive 19Fc expression by lentivirus in HL-
60 cells.
High quality protein was purified as shown by
immunoblotting with mAb KPL-1 and anti-
human IgG and silver staining of 19Fc.
19Fc bound on anti-human IgG polystyrene
beads were sheared with TRAP-6 activated
platelets in a cone-and-plate viscometer at
650/s. Binding was specifically blocked with
anti-PSGL-1mAb.
• MALDI analysis of O-glycans released from 19Fc
expressed in HL-60 cells, HEK293T and FUT7+HEK293Ts.
• Core-2 sLeX structure observed at m/z= 1879.7.
• Mono- and di-sialylated T-antigen structures at m/z=
895.1 and 1256.3, respectively
• Major (M) and minor (m) contributors to peaks were
identified using MALDI-TOF/TOF.
P-selectin
L-selectin
E-selectin
Confocal Microscopy Show ST6GalNAc2 and C2GnT-I Co-
localize
*P < 0.001 compared to control HL-60
0
20
40
60
80
100
0 1 2 3 4
HL60
ST6GalNAc1⁺
HL60 + KPL1
ST6GalNAc1⁺ + KPL1
HL-60-platelet
adhesion(%)
Time (min)
0
25
50
75
100
0 1 2 3 4
350 1/s
*
* * *
0
25
50
75
100
0 1 2 3 4
1200 1/s
* * *
0
25
50
75
100
0 1 2 3 4
3000 1/s
*
*
* *
0
25
50
75
100
0 1 2 3 4
4650 1/s*
* *
*
0 1 2 3 4
650 1/s
WT HL60
ST6GalNAc2⁺
Time (min)
HL-60-platelet
adhesion(%)
0
20
40
60
80
100
0 1 2 3 4
HL60
ST6GalNAc4⁺
HL60 + KPL1
ST6GalNAc4⁺ + KPL1
0
20
40
60
80
100
0 1 2 3 4
HL60
ST6GalNAc2⁺
HL60 + KPL1
ST6GalNAc2⁺ + KPL1
* *
*
*
*P < 0.05 compared to control HL-60
*P < 0.05 compared to control HL-60
*P < 0.001 compared to control HL-60
Scale bar = 20 μm
Objective and Methods
PSGL-1 variants similar to WT protein
β1,3
β1,4
β1,3
( )α2,3 1.9
2.8
36.3
6.4
45.3
3.5
3.9
14
72
N.R.
N.R.
N.R.
N.R.
β1,3β1,3
N.R.12
2
O-Glycan Structures
Aeed
et al.
Wilkins
et al.
Current
Study
α2,3
( )α2,3
α2,3
α2,3
α2,3
( )α2,3
( )α2,3
( )α2,3
( )α2,3
( )α2,3
( )α2,3
( )α2,3
GlcNAcGal FucSialic AcidGalNAc
N.R.
N.R.α2,3
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
15.1
35.3
3.4
16.8
11.7
0.7( )α2,3
HL60
PSGL-1
HL60
19Fc
β1,6
α1,3 α1,3 α1,3
β1,6
α1,3
β1,6
β1,3
β1,6
β1,4
β1,3
β1,3β1,4
α1,3
β1,6
β1,3
β1,4
α1,3
β1,6
β1,6
α1,3
α2,6
β1,3
α2,6
α2,6
β1,3α2,3
β1,3
β1,4
β1,3
β1,3β1,4
β1,3
β1,4 β1,4 β1,4
( )
β1,4β1,3β1,4β1,3β1,4
β1,4
β1,3
β1,4
α2,3
O-glycans of PSGL-1 quantified by stripping carbohydrates from either whole PSGL-1 by
Wilkins, P.P., et al. (J Biol Chem, 1996. 271(31): p. 18732-42) and Aeed, P.A., et al. (Glycoconj
J, 1998. 15(10): p. 975-85), or 19Fc (current manuscript). N.R. – Not Reported
Conclusion
ST6GalNAc-2 Over-expression Reduces Leukocyte Rolling on
P- and L-Selectin and Reduces Adhesion on E-selectin
 Competition between C2GnT-I and ST6GalNAc-2 may
be an important regulator of leukocyte selectin-binding
function