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Chemical & Biological Engineering and Clinical Translational
Research Center
State University of New York, Buffalo, NY, USA
Demystifying the Glycosciences• Glycobiology: Study of the function of sugars
attached to proteins and membranes, including protein- and lipid- linked sugars.
• Glycoconjugates: Formed when mono-, oligo- or poly-saccharides attach to proteins and lipids. This occurs in ER and Golgi (mostly).
• Glycans: Carbohydrate entity attached to proteins and lipids. Found outside cells, in cytoplasm (complex) and nucleus decorating transcription factors (simple).
• Lectins: Glycan binding proteins
Function of glycans• Structural component of cell wall and extracellular
matrix proteins [Cancer and stem cell biomarkers]• Intra- and extra-cellular trafficking of
glycoconjugates [Protein therapeutics half-life]• Cell adhesion: during cell-cell and cell-matrix
interaction. [Inflammation, human-virus and human-microbiome interactions]
• Cell signaling: Intracellular and extracellular [transcription factor regulation. O-GlcNAcformation on Ser/Thr competes with phosphorylation]
Why is glycosylation so complicated?
A. It is not part of the central dogma – not part of regular course work.
B. Because the biochemists made it look more complicated than it really is:
– Database lists 700 different monosaccharides. In humans there are only 9!
– They said that there are 1012 possible carbohydrates. In reality it is closer to 102-103 classical structures.
– The names are so complicated. Why doesn’t everyone just use IUPAC nomenclature
C. Because it is complicated
Common monosaccharides
• Most glycans are hexose sugars: 4 chiral carbons resulting in 16 different molecules (epimers and enantiomers).
• But many are of relative low abundance. Humans are made of 9 major monosaccharides.
All sugars are similar in structure
D-Glc (Glucose)D-GlcA
[oxidation of C6]
Xyl (Xylose)[remove C6]
D-Man (Mannose)[C2-epimerase]
D-GlcNAc[C2-acetylatedamino gp.]
D-Gal (Galactose)[C4-epimerase]
D-ManNAc
Sialic acid
+ pyruvate
L-fucose[6-deoxy-D-Gal]
D-GalNAc
O
OH
OH
OH
OH
OOH
OH
OH
OH
OH
OOH
OH
OH
OH
CH3
OOH
OH
NHAc
OH
OH
O
H
HH
H
OH
OH
H NHAc
OH
OH
O
OH
OHOH
OH
OH
O
OHOH
OH
OH
OOH
D-ManNAc
O
H
HH
H
OH
OH
H OH
OH
OH
1
2
34
5
9
7
6
8
Different families of glycans
Wiley Interdiscip Rev Syst Biol Med. 2015 Jul-Aug;7(4):163-81
Of course there are more sugars in plants and the microbiome…
http://www.ncbi.nlm.nih.gov/books/NBK310273/
… and more on bacteria that regulate immune function
A. TEM of T. forsythia showing glycans. B. Schematic O-glycan core to protein. Terminal trisaccharide is circled. C. Immune signaling by C-type lectin-like receptor (CLR) and TLR2 activated by O-glycans and TLR2 ligands (e.g., BspA) orchestrates.
Front. Microbiol., 17 October 2013
Relevance to human diseases
• Human glycans bind bacterial lectin-like adhesins
• Bacterial carbohydrates bind mammalian lectins
• Bacterial carbohydrates mimic human glycans, and condition the microbiome
Fusobacterium nucleatum• Plays a role in periodontal disease.• Associated with colon cancer
wikipedia
Plasma Membrane
Roseman, S. J. Biol. Chem. 2001;276:41527-41542
Glycocalyx – Physically big
Translated protein
Post-translational modification
Functionalprotein
Ribosome
ER/Golgi
Cell-surface
Glycosyltransferases
Neelamegham and Mahal, Current Opinion of Structural Biology, 2016
Lectins: Glycan binding proteinsC-type lectins (selectins)
Cell. 2000 Oct 27;103(3):467-79.
Siglecs• Contains
shallow Argthat binds sialicacid
Schnaar, J. Leuk. Biol, June, 2016
Galectins
Nature Reviews Microbiology 7, 424-438 (June 2009)
Systems Glycobiology
?
Input
Decorated GlycoProbes
GlycoProbes
Output
WT or edited/knock-out cell
Cell rolling or adhesion phenotype
GNAT(Glycosylation Network
Analysis Toolbox)
GlycoPAT(GlycoProteomicsAnalysis Toolbox)
http://sourceforge.net/projects/gnatmatlab/
Smallmolecules
ST3Gal-3
ST3Gal-4
ST3Gal-6 α(1,3)FUT
β3
β6
α3
β4α
β4 α3
β3
α3
αβ6 β4 α3
α3
β3
α3
α β6
Type II α(2,3) Sialyllactosamine
sialyl LewisX
Which α(2,3)sialyltransferase, ST3Gal-3, -4 or -6, contributes to human selectin-ligand
biosynthesis?
Sialic acid
Galactose
Mannose
GlcNAc
GalNAc
Fucose
Mono.Sugar
Alexander Buffone Nandini Mondal
Mondal et al. Blood. 125:687-96, 2015.
Wild-Type HL-60
ST3Gal-3‾HL-60
ST3Gal-4‾HL-60
ST3Gal-6‾HL-60
ST3Gal-3‾4‾HL-60
ST3Gal-4‾6‾HL-60
ST
3Gal
-3
ST
3Gal
-4
ST
3Gal
-6
β-a
ctin
Making ST3Gal ‾ HL60 cells
Single knock down cells
ST3Gal-3 ͞
ST3Gal-4 ͞
ST3Gal-6 ͞
Dual knock down cells
ST3Gal-3 ͞ 4 ͞
ST3Gal-4 ͞ 6 ͞
5’LTR U6ST3GalshRNA
DsRed 3’LTRPGKVector-3
Buffone et al. J Biol Chem. 288(3):1620-33, 2013.
HL60 cell rolling on E-Selectin
A B
C D
Genome Editing with CRISPR-Cas9
The Scientist, 2014
HL-60 ST3Gal-4 knock out generated using CRISPR/Cas9
WT 51400 5’-GGGTAG…ATCTTCCTGCGGCTTGAGGATTATTTCTGGGTC–3’51622
KO 51400 5’-GGGTAG…ATCTT-----------------ATTTCTGGGTC–3’51622
KO 51400 5’-GGGT------238bp-------GAGGATTATTTCTGGGTC-3’51622
Guide
RNA NLS NLShSpCas9 bGHpAU6 CBh
β4 α3
α3
β3
α3
α β6
β4
β4
α3
100 101 102 103 104100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
Co
un
t
100
75
50
25
0
LeXsLeXHECA-452 ECL lectin
Abrogation of leukocyte rolling on all selectins
0
50
100
150
WT
P2
H3
ST
3G
al-4
-KOIn
tera
cti
ng
cell
s/m
m2
†‡†‡
WT
ST
3G
al-4
-KO
*
**
WT
ST
3G
al-4
-KO
AdherentRolling
*
**
E-selectin L-selectinP-selectin
0
25
50
75
100
0 10 20 30
Ro
llin
g c
ell
s (
%)
Rolling velocity (μm/sec)
WT (9/160)ST3Gal-4-KO (10/85)
Profiling glycans
N-glycans: ST3Gal-4 controls sLeX biosynthesis
Sialic acid
Galactose
Mannose
GlcNAc
GalNAc
Fucose
Mono.Sugar
WT HL-60
ST3Gal-4 KO HL-60
O-glycans: ST3Gal-4 deletion abrogates sLeX biosynthesis
Sialic acid
Galactose
Mannose
GlcNAc
GalNAc
Fucose
Mono.Sugar
Human neutrophils derived from hematopoietic stem cells (HSCs)
57% 43%
ST3Gal-4¯hHSc derived
neutrophils
DsRed Expression
0
20
40
60
80
100
120
0 2 4 6 8 10 12
Recep
tor
exp
ressio
n (
MF
I)
Time (Day)
CD11bCD34
100 101 102 103 104
1024
754
512
252
0
Fo
rward
scatt
er
5’LTR U6ST3GalshRNA
DsRed 3’LTRPGKVector
Human neutrophil rolling on selectins
0
100
200
300
400
500
DsR
ed
ST
3G
al-4‾In
tera
cti
ng
cell
s/m
m2
*
**
0
100
200
300
400
500
600
700
DsR
ed
ST
3G
al-4‾
*
0
50
100
150
200
250
300
DsR
ed
ST
3G
al-4‾
AdherentRolling
*
L-selectinP-selectinE-selectin
DsRed+ cells(stimulated HUVECs)
The multistep cell adhesion cascade
Mac-1 (αM:β2)Chemokine
receptor
LFA-1 (αL:β2)
E-selectin
Chemokine (IL-8)
ICAM-1
CD31
P-selectin
Blood flow
Endothelium
1. Tethering/Capture 2. Rolling 3. Activation 4. Firm adhesion & diapedesis
Rapid slow
Glycan dependent Glycan independent
L-selectin
Blood. 125(4):687-96, 2015ST3Gal-4 dependent
Rapid glycan profiling and chemical synthesis
Nature Methods 13, 81–86 (2016)
The multistep cell adhesion cascade
Mac-1 (αM:β2)Chemokine
receptor
LFA-1 (αL:β2)
E-selectin
Chemokine (IL-8)
ICAM-1
CD31
P-selectin
Blood flow
Endothelium
1. Tethering/Capture 2. Rolling 3. Activation 4. Firm adhesion & diapedesis
Rapid slow
L-selectin
ST3Gal-4 dependentGlycolipids Glycolipids/N-glycans
Glycan dependent
O-glycans
N-glycans
GlycoPAT: High-throughput glycoproteomics analysis
Tandem mass spectrometry in Orbitrap
Protease
Digestion
(Trypsin,
Glu-C etc.) nanoLC
MS1
Separation based on
precursor m/z
MS2
fragmentationm/z
Inte
nsi
ty
Sialic acid
Galactose
Mannose
GlcNAc
GalNAc
Fucose
Mono.Sugar
With Jun Qu
Digest in silicoCandidate glycopeptide list
In SmallGlyPep format
Protein list
Non-glycan PTM list
Digesting protease
3
4
5
Overall algorithm
List of potential glycans
Enzymes
in system
Some of the
expected glycans
Connection Inference
using
GNAT
1 2
GlycoPAT: High-throughput glycoproteomics analysis
Overall algorithm
List of potential glycans
Enzymes
in system
Some of the
expected glycans
Connection Inference
using
GNAT
12
Digest in silicoCandidate glycopeptide list
In SmallGlyPep format
Protein list
Non-glycan PTM list
Digesting protease
34
5
MS1 match
within tolerance?
6 Tandem MS
expt. data
Experimental
[M+H]Theoretical
[M+H]
MSn Spectra scoring
Xcorr
P-value
% Ion match
Top10 peaks
Ensemble & False Discovery
Score (ES) Rate (FDR)
MSn frag. spectra
Theoretical
MSn
Spectra
yes
GlycoPAT: High-throughput glycoproteomics analysis
• Xcorr• P-value• Top10• % ion-match
Ensemble score
Scoring parameters
GlycoPAT: High-throughput glycoproteomics analysis
Header
Scoring Table
FDR
options
Characterization of:
• Simple standard proteins
• Mixtures of standards
• Plasma cryoprecipitate from 5cc of blood
Cell Adhesion GlycoEngineering
• MSCs (Mesenchymal stem
cells) and CDCs (Cardiosphere
derived stem cells) are
promising cellular therapuetics.
• However, promising results
from animal studies are not
translating to clinical benefits
• Local infusion to damaged
tissue is not beneficial
• Systemic infusion proximal
to the therapeutic site
– minimally invasive
– allows repeated treatmentStrauer B E , and Kornowski R Circulation. 2003;107:929-934
Goals• Make stem cells home to sites of
inflammation, much like white blood cells
• Create methods that can be used in a clinical setting with minimal effort
• Move studies to clinically relevant large animals
Chi Lo
Experimental plan
Test modified cell binding to selectins
in vitro
Check whether treatment increases
homing in vivo
Increase α(1,3) fucose
Coupling rPSGL-1 –19Fc[FUT7+]
Adding scaffold and
sugar
Adding sugar
Thr-57}
Tyrosine
sulfationTransmembrane
domain
Decamer
Repeats
O-glycanN-glycan
Cytoplasmic
domain42 118 279 321 341 412
PSGL-1
Sialyl Lewis-x
NeuAcGal GlcNAc
Fuc
Recombinant PSGL-1
19 aa
PSGL-1
Human
IgG1
19Fc [FUT7+]
Characterization of 19Fc
19Fc
Mass (m/z)
% I
nte
ns
ity
19Fc[FUT7+]
Mass (m/z)%
In
ten
sit
y
Lo, C.Y., et al. Biomaterials, 2013. 34(33): p. 8213-22.
Coupling recombinant PSGL-1
Palmitate
Protein G (PPG)
+ +
19Fc or
19Fc[FUT7+]
MSC or CDC
CDC MSC
30 min. 30 min.
19Fc coupling with PPG increases
interactions with P-selectin
*
**
**
*
Experimental plan
Test modified cell binding to selectins
in vitro
Check whether treatment increases
homing in vivo
Increase α(1,3) fucose
Coupling rPSGL-1 –19Fc[FUT7+]
Adding scaffold and
sugar
Adding sugar
Over-expression of FUT7
Lentiviral constructs
CMV R U5
5’ LTR 3’ LTR
U3EGFPCMV FUT7 R U5
Fucose
Sialyl Lewis-x
NeuAc Gal GlcNAc
Fuc
FUT7 increases CDC
interactions with
stimulated HUVECs
CDC CDC FUT7
CDC FUT7 +
Esel blocking
Large animal studies: Swine
Test modified cell binding to selectins
in vitro
Check whether treatment increases
homing in vivo
Increase α(1,3) fucose
Coupling rPSGL-1 –19Fc[FUT7+]
Adding scaffold and
sugar
Adding sugar
Pig experiments: 30 min. brief ischemia-
reperfusion of LAD
Catheter placement
for cell injection
echocardiogram
CD
C-F
UT7
CD
C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Co
pie
s o
f F
UT
7/S
RY
+ c
ell
0
10
20
30
40
50
60
70
80
90
100 ×103
Left anterior
descending (LAD)
region
A B C
To
tal
fun
cti
on
ali
ze
d c
ell
s r
eta
ine
d
/g t
iss
ue
0
5
10
15
20
25
1 2 3
Avg
# F
UT
7 g
en
e c
op
ies
/10
0 n
g f
em
ale
ge
no
mic
DN
A
Regions:
*
Avg
# F
UT
7 g
en
e c
op
ies
/10
0 n
g f
em
ale
ge
no
mic
DN
A
Co
pie
s o
f F
UT
7/S
RY
+ c
ell
Pig experiments: 30 min. brief
ischemia-reperfusion of LAD
CD
C-F
UT7
CD
C
Lo, C.Y. et al. Biomaterials (2016), 74: 19-30
Conclusion• Glycosylation is a poorly communicated field--
its easier than it looks
• There are tremendous opportunities for exploration:
– New basic science
– New applications
– New drugs
The final frontier--- boldly go where no (wo)man has gone before
AcknowledgementsCurrent lab members:
Anju Kelkar, Ph.D. Arezoo Momeni
Kai Cheng Changjie Zhang
Hannah Wang Yuqi Zhu
Xinheng Yu Yusen Zou
Collaborators:
Aristotelis Antonopoulos, Anne Dell and Stuart Haslam: Imperial College-London
S. G. Sampthkumar: National Institute of Immunology (India)
John Canty: Cardiology, Univ. at Buffalo
Joseph Lau, Khushi L. Matta: Roswell Park Cancer Institute, Buffalo, NY
Funding support:
NIH Heart, Lung and Blood Institute: Systems Biology program
American Heart Association
NY State Stem Cell Program