Download Characterizing the O-glycosylation landscape of human plasma

REGULAR ARTICLE
Characterizing the O-glycosylation landscape of human plasma, platelets,
and endothelial cells
Sarah L. King,1 Hiren J. Joshi,1 Katrine T. Schjoldager,1 Adnan Halim,1 Thomas D. Madsen,1 Morten H. Dziegiel,2 Anders Woetmann,3
Sergey Y. Vakhrushev,1 and Hans H. Wandall1
1
Department of Cellular and Molecular Medicine, Centre for Glycomics, University of Copenhagen, Copenhagen, Denmark; 2Department of Clinical Immunology,
Section 2034, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark; and 3Department of Immunology and Microbiology, University of Copenhagen,
Copenhagen, Denmark
Key Points
• Human platelets, endothelial cells, and plasma
proteins are extensively
O-glycosylated, with
.1123 O-glycosites
identified in this study.
• O-glycosites can be
classified into functional subgroups; one
important function includes the protection
from proteolytic
processing.
The hemostatic system comprises platelet aggregation, coagulation, and fibrinolysis, and is
critical to the maintenance of vascular integrity. Multiple studies indicate that glycans play
important roles in the hemostatic system; however, most investigations have focused on
N-glycans because of the complexity of O-glycan analysis. Here we performed the first
systematic analysis of native-O-glycosylation using lectin affinity chromatography coupled
to liquid chromatography mass spectrometry (LC-MS)/MS to determine the precise location
of O-glycans in human plasma, platelets, and endothelial cells, which coordinately regulate
hemostasis. We identified the hitherto largest O-glycoproteome from native tissue with
a total of 649 glycoproteins and 1123 nonambiguous O-glycosites, demonstrating that
O-glycosylation is a ubiquitous modification of extracellular proteins. Investigation of the
general properties of O-glycosylation established that it is a heterogeneous modification,
frequently occurring at low density within disordered regions in a cell-dependent manner.
Using an unbiased screen to identify associations between O-glycosites and protein
annotations we found that O-glycans were over-represented close (6 15 amino acids) to
tandem repeat regions, protease cleavage sites, within propeptides, and located on a select
group of protein domains. The importance of O-glycosites in proximity to proteolytic
cleavage sites was further supported by in vitro peptide assays demonstrating that
proteolysis of key hemostatic proteins can be inhibited by the presence of O-glycans.
Collectively, these data illustrate the global properties of native O-glycosylation and provide
the requisite roadmap for future biomarker and structure-function studies.
Introduction
Mucin-type O-glycosylation, or N-Acetylgalactosamine (GalNAc) O-glycosylation is arguably the most
prevalent and diverse form of O-glycosylation.1 GalNAc O-glycosylation (hereafter O-glycosylation)
biosynthesis is initiated by a family of as many as 20 differentially expressed GalNAc-transferases (GalNAcTs)2 that add a GalNAc monosaccharide to selected serine (Ser), threonine (Thr), and, possibly, tyrosine
(Tyr) residues.1,3 The major O-glycan structures in humans are the sialylated Core 1 O-glycans—sialyl T and
disialyl-T—along with the lesser represented Core 2 glycans (Figure 1).4,5 These can be further elongated
and modified, generating a large set of O-glycan structures.4-6 Historically, O-glycosylation has been
considered a relatively rare, densely clustered, posttranslational modification (PTM) occurring in mucins
and mucin-like proteins. Such O-glycosylated regions are thought to attract water molecules, stabilize
Submitted 13 October 2016; accepted 17 January 2017. DOI 10.1182/
bloodadvances.2016002121.
© 2017 by The American Society of Hematology
The full-text version of this article contains a data supplement.
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
429
3
GalNActransferases
(x20)
Sialic acid (Neu5Ac)
3
PN
A
VVA
Fucose (Fuc)
Galactose (Gal)
N-acetylgalactosamine (GalNAc) Ser/Thr
(Tyr)
N-acetylglucosamine (GlcNAc)
Tn
3
3
3
6
Ser/Thr
(Tyr)
disialyl-T
Ser/Thr
(Tyr)
T
Glycan binding protein (lectin)
3
3
Core 1
3 3
3
6
3
Ser/Thr
(Tyr)
4
SO33
4
6
Ser/Thr
(Tyr)
complex Core 2
Core 2
Figure 1. Major O-glycan structures in human serum and endothelial cells. The majority of O-glycans in human serum4 and HUVECs5 are formed from sialylated Core 1
(Galb1-3GalNAcaSer/Thr) and Core 2 (GlcNAcb1-6[Galb1-3] GalNAcaSer/Thr) structures. Mature glycans are capped with variable numbers of negatively charged sialic acid
(Neu5Ac), which can be removed enzymatically by neuraminidases. O-glycans can also be found in fucosylated and sulfated forms. An example of a complex Core 2 O-glycan
carrying a 6-O-sulfated sialyl-Lewis x terminal structure is shown at right. The lectin enrichment strategy employed in this study uses VVA and PNA to predominantly target the
biosynthetic intermediate, Tn, and Core 1 O-glycans.
protein structure, extend the polypeptide backbone, and protect from
proteolytic cleavage.7 Recent glycoproteomics studies in cell lines,
however, indicate that, far from being specific to mucin-like regions,
O-glycosylation is a ubiquitous PTM and .80% of proteins trafficking
through the secretory pathway are estimated to be O-glycosylated.1,8
Furthermore, it is likely that O-glycans play a role in diverse physiologic
systems because altered O-glycosylation is associated with IgA
nephropathy,9 Tn syndrome,10 Crohn disease,11 tumorigenesis,12
impaired leukocyte recruitment,13 and high-density lipoprotein levels.14
Dissection of the molecular mechanisms by which O-glycans affect
these systems is, however, currently impeded by a lack of knowledge of
specific O-glycan sites in vivo.
The hemostatic system comprises platelet aggregation, coagulation,
and fibrinolysis and is critical for the maintenance of vascular integrity.
Within the hemostatic system, the presence of glycans on individual
proteins has been demonstrated to alter expression, clearance, and
catalytic activity.15 O-glycans on von Willebrand factor (VWF) have
been associated with changes in VWF plasma concentration,16
platelet binding,17 and response to shear stress.18 O-glycosylation is
also required for P-selectin–dependent leukocyte rolling,19,20 and
O-glycosylation of platelet glycoprotein Ib a (GP1BA) is important for
binding to VWF.21 Similarly, coagulation factors have been reported
to be modified by O-glycosylation. Activation of coagulation factor X
(FX) by Russell’s viper venom and Xase is altered in the presence of
O-glycans,22 as is the sensitivity of coagulation factor XII (FXII) to
contact activation, with loss of FXII O-glycans recently implicated in
the pathogenesis of hereditary angioedema.23 Furthermore, murine
studies have shown that O-glycans are important for the hemostatic
process in vivo, because truncation of O-glycans results in severe
bleeding deficits, aberrant angiogenesis, and platelet biogenesis,
whereas loss of specific O-glycan sites leads to increased bleeding
time and VWF insufficiency.24-26
These examples suggest important functions of O-glycosylation, yet,
primarily because of technical limitations, we have little information on
where the O-glycans are localized. Unlike N-linked glycosylation, no
consensus sequence has been found for O-linked glycosylation, which
further complicates analysis and prediction of O-glycan sites. Recent
developments in mass spectrometry and glycan-enrichment methods,
however, have enabled global studies of site-specific glycosylation
using native tissues.8,27-30 Such glycoproteomic analyses have been
430
KING et al
applied to identify O-glycosites in human cerebrospinal fluid28 and
urine.29 More recently, substantial progress has been made in the
analysis of human plasma and serum,30,31 although the overall number
of glycopeptides identified has been modest because of difficulties
achieving high-sensitivity glycopeptide enrichment.
Here we extend this knowledge using a dual vicia villosa agglutinin
(VVA) and peanut agglutinin (PNA) lectin weak affinity chromatography (LWAC) enrichment strategy1,8,32 coupled to higher-energy
collisional dissociation (HCD) and electron transfer dissociation
(ETD) liquid chromatography mass spectrometry (LC-MS) and present
the first large-scale identification of O-glycosites in platelets, plasma,
and endothelial cells.
Materials and methods
For a more detailed methods section see the supplemental Materials.
Glycoproteomic analysis
AB RhD–positive platelets from 4 random donors and plasma were obtained
from the Blood Bank of the Capital Region and harvested according to
standard protocols. Primary Human Umbilical Vein Endothelial Cells
(HUVEC) were purchased from Life Technologies. 0.5 mL of packed cells
or 2 mL plasma was prepared for O-glycoproteomic analysis as previously
described.33,34 Briefly, samples were sonicated, de-sialylated with 0.1 U/mL
Clostridium perfringens neuraminidase Type VI (Sigma) for 1 hour at 37°C,
reduced (5 mM dithiothreitol, 60°C, 30 minutes), alkylated (10 mM
iodoacetamide, room temperature, 30 minutes), and subjected to overnight
digestion with MS-grade trypsin (Roche) or chymotrypsin (Roche) at
1:50 wt/wt. Samples were then purified on a C18 SepPak (Waters) and
dried by SpeedVac. Glycopeptides were enriched by lectin chromatography using PNA- or VVA-agarose (VectorLabs) on an AKTA fast protein
liquid chromatography system. Eluted glycopeptides were then de-salted
using C18 Stage Tips before Orbitrap LC-MS/MS analysis. MS/MS
spectra were interrogated against the nonredundant human proteome
using the SEQUEST-HT search engine in Proteome Discoverer. These
data can be fully interrogated online (http://glycodomain.glycomics.ku.dk/
doi/10.1182/bloodadvances.2016002121/) using the Glycodomain Viewer,1
and mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE35 partner repository with the
dataset identifier PXD004590.
Bioinformatics
All bioinformatics and statistical analyses were performed using R statistical software. Where required, data were compared with a background
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
B
A
endothelial
cells
Sample
Preparation
HUVEC
(359)
214
35
plasma
platelets
153
1. reduction & alkylation
34 76
57
3. digestion
25 105
19
256 54 209
80
plasma
(279)
2. de-sialylation
HUVEC
(604)
455
platelet
(247)
plasma
(354)
O-glycoproteins
n= 649
platelet
(387)
O-glycosites
n= 1123
C
complex peptide
mixture
this study
((649)
541
Lectin Weak Affiinity
Chromatography
this study
(649)
231
418
LWAC
655
108
published
(161)
53
O-glycoproteins
(native)
LWAC
glycopeptides
published
(1073)
O-glycoproteins
(total)
D
Intensity
FT-MS
ETD-MS2
HCD-MS2
m/z
n=159
n=3412
0.02
*
*
0.00
ms/ms
1e+03
1e+05
1e+03
1e+05
Protein copy number
* proteins below LOQ
Retention time
E
F
800
710
fibrinogen alpha
600
200
Protein count
Proteome
Discoverer,
R
Glycosite count
Identification and
Mapping
Proteome
0.04
Protein number
(rel. density)
Relative abundance
LC-MS/MS
O-glycoproteome
398
400
fibronectin
100
200
15
0
0
S
T
Y
Amino acid linkage
0 10 20 30 40 50
Glycans per protein
Figure 2. Enrichment and identification of glycopeptides in the hemostatic system. (A) Depiction of the proteomics workflow. Human platelet, plasma, and endothelial
samples were reduced, alkylated, de-sialylated using neuraminidase, and subsequently digested using either chymotrypsin or trypsin. Glycopeptides were then enriched from the resulting
complex peptide mixtures by sequential LWAC using VVA and PNA lectins. VVA enrichment was not used for plasma samples because of the absence of Tn glycans; however, plasma
samples were further separated using isoelectric focusing to reduce sample complexity. Fractions containing glycopeptides were separated by online reverse-phase liquid chromatography
followed by identification using Orbitrap FTMS. (B) Glycoproteins and glycosites identified in each sample in this study. (C) Overlap of the O-glycoproteins identified in this study with
previously published O-glycoproteins. Left, O-glycoproteins identified in native samples. Right, overlap with all reported O-glycoproteins. (D) Sensitivity of LWAC detection. The abundance
of each platelet O-glycoprotein was determined based on the platelet protein copy numbers reported in Burkhart et al36 and plotted as a histogram (left) alongside the total platelet protein
abundance obtained from the same study (right). Proteins with ,500 copies are below the limit of quantification and are indicated by an asterisk. Comparison of the 2 distributions indicates
that O-glycoproteins are detected over the full dynamic range of protein expression and include a similar proportion of proteins that are present below the limit of quantification. Note that
membrane proteins are not quantified and therefore excluded from analysis. (E) Count of Ser-, Thr-, and Tyr-linked O-glycans. (F) Number of unambiguous glycosites per protein.
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
O-GLYCOSYLATION IN THE HEMOSTATIC SYSTEM
431
hemostatic proteome.36-38 Data sets used during the analysis are listed in
supplemental Table 1. Proportional Venn Diagrams were generated using
BioVenn.39 GO term analysis was performed using the DAVID bioinformatics resource (https://david.ncifcrf.gov/).40 Only GO terms demonstrating
a twofold or greater change and represented by $10 proteins with a
PBenjamini , .001 were considered.
Synthesis of glycopeptide substrates
Synthetic 20-mer peptides (NEO biolabs) were designed around known
glycosylation sites and subjected to in vitro glycosylation using recombinant
glycosyltransferases expressed as soluble secreted truncated proteins in
insect cells, and were purified as described previously.41 10 mg of acceptor
peptides and 2 mM uridine triphosphate–GalNAc as the sugar donor were
prepared in 25 mL buffer containing 25 mM cacodylic acid sodium pH 7.4,
10 mM MnCl2, 0.25% Triton X-100. After 4- and 16-hour incubations at
37°C, reaction products were analyzed by MALDI-TOF-MS to determine
glycosyltransferase specificity. For cleavage assays, 50 mg of peptide was
glycosylated as described before, acidified, and purified by ultra-highperformance liquid chromatography on a C18 column. Control peptides
were subjected to the same process in the absence of donor sugar.
In vitro cleavage assays
1.25 nmol (100 mM) glycosylated or control peptides were incubated for the
indicated times with 50 nM plasmin (Sigma) or 50 nM human thrombin
(Sigma) in 100 mM Tris-HCl, pH 7.4, 100 mM NaCl; 500 nM coagulation
factor Xa (Haematologic Technologies) in 40 mM Tris-HCl, pH 8, 100 mM
NaCl, 2 mM CaCl2; 50 nM neutrophil elastase (ENZO Life Science) in
50 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid (HEPES),
pH 7.4, 150 mM NaCl, 0.05% Brij-35; 50 nM MMP7 or 100 nM MMP12
(R&D Systems) in 50 mM Tris-HCl, pH 7.4, 10 mM CaCl2, 150 mM NaCl,
0.05% Brij-35, 0.01 mM ZnCl2. Both MMP7 and MMP12 were activated with
1 mM AMPA for 30 minutes at 37°C before assay.
Results
Human platelet, plasma, and endothelial cell proteins
are extensively O-glycosylated
To identify which proteins are O-glycosylated and where the Oglycans are localized, we used our 2-step lectin enrichment strategy
coupled to LC-MS/MS to analyze the global native O-glycoproteome.
Enrichment of glycopeptides is essential for glycan detection in
complex samples, and the strategy to identify O-glycosylation sites
is shown in Figure 2A. We first performed lectin profiling of
human platelets, plasma, and endothelial cells and demonstrated
that all samples predominantly expressed sialylated Core 1 O-glycan
structures (sialyl-T; supplemental Figure 1). A small amount of the
nonsialylated Core 1 structure (T) was detected in nontreated
endothelial cells, but not in platelet or plasma samples. Endothelial
and, to a much lower degree, platelet samples also demonstrated
weak expression of the nonsialylated biosynthetic intermediate, Tn, on
a small subset of proteins. To identify specific sites of O-glycosylation,
de-sialylated samples were enriched by LWAC using PNA and VVA
lectins, which recognize T and Tn structures, respectively. Glycopeptides were subsequently identified using HCD and ETD LC-MS/MS
(Figure 2A; supplemental Tables 2-5). Using this approach, a total of
649 unique O-glycoproteins were detected (Figure 2B). On these
glycoproteins, 1123 O-glycosites could be unambiguously assigned.
A further 547 ambiguous O-glycopeptides carrying 700 glycans were
identified in which the glycan position could not be confidently
assigned within the peptide because of insufficient MS2 fragmentation to report the corresponding diagnostic fragment ions or missing
ETD spectra. In total, 1848 O-glycans were identified. These data
432
KING et al
represent a substantial increase in the existing knowledge
(Figure 2C). Because mass spectrometric analysis of native samples
such as platelet lysates is challenging due to the saturation of signals
by highly abundant proteins, we compared the dynamic range of the
platelet O-glycoproteome to the published quantitative proteome36
to better understand the depth of coverage achieved. Even in the
absence of extensive precolumn separation, we found the range of
glycoprotein detection to recapitulate that of total protein expression.
With glycopeptide enrichment, low copy number (,500) proteins
were detected, demonstrating that the method can be applied to
complex native samples (Figure 2D).
We next sought to define the general properties of native
O-glycosylation using a bioinformatic analysis of the location,
distribution, and clustering of identified O-glycosites. As previously
described in cell studies,1 we found Thr is preferentially glycosylated over Ser, whereas Tyr is only rarely glycosylated (Figure 2E). In
contrast to the canonical view of O-glycosylation as a high-density
mucin-type modification, but in line with previous findings in cell
lines,1 76% of glycoproteins identified in the present study carried
#3 sites of O-glycosylation (Figure 2F). Only a small proportion (28
proteins, 4.3%) were found to be highly glycosylated (.10 sites),
with fibrinogen a and fibronectin the most highly glycosylated
proteins identified with .30 sites each. Glycosylation was also
found to be heterogeneous because analysis of overlapping,
multisite peptides indicated that O-glycosylation often occurred
at substoichiometric levels (supplemental Figure 2A). Similarly,
although the majority of glycans identified were T rather than Tn
structures (supplemental Figure 2B), there was considerable
structural heterogeneity at individual sites (supplemental Figure 2C).
Interestingly, when analyzing the distribution of identified glycan
structures, proteins carrying solely Tn-glycans were highly enriched
for the annotation endoplasmatic reticulum, potentially indicative of
a discrete functional role for these glycoproteins (supplemental
Figure 2B, inset).
Previously it has been reported that GP1BA,42 P-selectin
glycoprotein ligand 1 (PSGL1)43,44 and VWF,45 among others,
carry Core 2 structures and that these structures make up ;2%
of the plasma O-glycoproteome.5 Enrichment and MS analysis
of these structures is problematic, and only a handful of sites
carrying complex O-glycans have been identified using
glycoproteomics.29,46,47 We reasoned, however, that a subset of
these sites may be detectable because of co-elution with T/Tn
peptides in our study. We therefore developed a script to search for
spectra containing [HexNAc2]1 (m/z 407) and [HexHex-NAc2]1
(m/z 569) diagnostic peaks to determine whether there was
evidence of branched O-glycans in our data set. In addition, we
applied the algorithm described by Halim et al to predict the
presence of GlcNAc monosaccharides.48 Combining these data,
we found spectra representing branched, possible core 2,
structures on 64 (9.9%) proteins. Glycosites predicted to carry
these structures are highlighted in supplemental Tables 2-5 and
supplemental Figure 3.
The majority of O-glycosites discovered to date have been
identified in immortalized cell lines that have been glycoengineered to homogeneously express the truncated Tn antigen.1
Because the effects of O-glycan truncation on O-glycan location
are unknown, it is not clear whether previously identified sites are
representative of native glycosylation. We therefore compared
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
O-GalNAc in vivo
vit. K dependent protein S (2)
O-GalNAc recombinant/cells
tissue factor pathway inhibitor (6)
fibronectin
prothrombin (5)
EGF/EGF-like
plasminogen (4)
kringle/kringle-like
urokinase (2)
peptidase
laminin G
tissue-type plasminogen activator (1)
plastocyanin
carboxypeptidase B2 (1)
sushi
vit. K dependent protein C (1)
cystatin
serpin
coagulation factor IX (6)
cysteine knot
coagulation factor X (4)
WVFD
coagulation factor XII (7)
WVFA
WVFC
coagulation factor XIIIB (1)
TIL
kininogen-1 (18)
51x
coagulation factor V (53)
activation peptide
coagulation factor VIII (8)
activation peptide
plasminogen activator inhibitor 1 (2)
plasma serine protease inhibitor (4)
plasma protease C1 inhibitor (10)
alpha-1-antitrypsin (6)
VWF (11)
Figure 3. Summary of total O-glycosylation of coagulation factors and inhibitors. Protein schematics illustrating the location of O-glycosites relative to Uniprot domains on
coagulation factors and inhibitors. The total number of unambiguous sites is indicated in brackets on the right. Sites identified in native (“in vivo”) samples in either this study or prior
publications are indicated by a yellow square. Sites identified on recombinant proteins or in immortalized cell lines are indicated by a stroke. Many sites are located at the N-termini of
the protein, or within processed regions (either propeptides, regions cleaved for activation, or on activation peptides). This is exemplified by the extensive glycosylation of the
activation peptide of FV, but is also evident on the majority of proteins shown above. EGF, epidermal growth factor; TIL, trypsin inhibitor-like.
sites identified here with previously published sites to determine
whether general properties of O-glycosylation were conserved
between glycoengineered cells and native samples. No substantial differences were noted. Glycosites were found to reside in
disordered regions within the extracellular or lumenal segments
of proteins (supplemental Figure 4A). The majority of glycans
occurred as individual sites as previously described49; however,
where multiple sites were identified, they were found to weakly
cluster (supplemental Figure 4B). No clear consensus sequence
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
could be defined for O-glycans, although the amino acids Pro, Ser,
and Thr were over-represented, and negatively charged amino
acids were under-represented around glycosylation sites relative
to randomly sampled Ser/Thr (supplemental Figure 4C). Glycosites were found along the whole length of proteins, with a small
but significant enrichment in the N-terminal (at 40-70 amino acids;
supplemental Figure 5). Taken together, these data demonstrate
that O-glycosylation is a ubiquitous modification of disordered
regions in the extracellular space. Furthermore, the site
O-GLYCOSYLATION IN THE HEMOSTATIC SYSTEM
433
A Platelet Receptors
O-GalNAc in vivo
O-GalNAc recombinant/cells
VWF
fibrinogen, fn
collagen
VWF
collagen
*
-S-S-
GPIX
GPIBb
GPV
GPIBa
αIIb
β1
α2
β3
(Glycoprotein IIb/IIIa) (Glycoprotein Ia/IIa)
GPVI
B
Fibronectin
7x
fibrin, fn
10x
collagen
fn
fn
C
integrin
7x 4x 11x 5x
6x
fibrin
syndecan
heparin
fn
α
Fibrinogen
)
D
β 231
γ
179
disulfide
rings
x 65
19
52-6
(2
223
180
161
123
coiled-coil
D
623
45
106
64
47 66
35 44
coiled-coil
E
Figure 4. O-glycosylation of platelet receptors, fibronectin, and fibrinogen. (A) Platelet receptors. Platelet receptors are critical mediators of platelet activation and
adherence, and include the well-described GPIB-V-IX complex, the collagen binding GPVI, and multiple integrin receptors.50 GPIB a is part of the GPIB-V-IX (VWF receptor)
complex and contains a mucin-like macroglycopeptide stem region, which is known to be O-glycosylated; however, the specific sites are poorly described, with only a single probable
site at Thr308 (indicated by and asterisk).51 In this study, 7 glycosites were identified in this region. Novel O-glycosylation was identified on all other members of the complex in regions
flanking the leucine-rich repeats. An additional novel O-glycosite was also detected on the collagen receptor GPVI. Integrin receptors (glycoproteins IIb/IIIa and Ia/IIa) were also
found to be O-glycosylated at multiple sites. In particular, novel glycosylation was identified in the VWFA domains of b integrins (b1 and b3), and a novel glycosite was identified
juxtaposed to the transmembrane region of integrin a2. Major ligands for each of the receptors are indicated above the receptors. (B) Fibronectin. Four sites of O-glycosylation have
been described for fibronectin, 3 sites located in the variable region (T2024, T2064, T2065) and a fourth site in an N-terminal linker region (T279). All sites except T2024
were identified in this study. Moreover, in total, hemostatic fibronectin was found to carry 31 unambiguous glycosites and 14 additional ambiguous sites of glycosylation. In total,
71 unambiguous sites have been identified on fibronectin from all sources. (C) Fibrinogen. Fibrinogen a was found to be extensively glycosylated, with 45 glycosites identified
in hemostatic samples and 68 sites identified across all sources. The glycosites were distributed across the protein, with the majority located within the coiled-coiled domain.
Fibrinogen b and g were much less glycosylated and were found to carry just 2 and 1 sites in this study, respectively. In total, across all studies, 4 sites have been detected on
fibrinogen b and 7 on fibrinogen g. Because of the large number of identified sites, no distinction is made between native (in vivo) and in vitro glycosylation on fibronectin and
fibrinogen. Fibronectin (fn).
distribution and localization properties between hemostatic native
tissues and immortalized cells are conserved.
Site-specific O-glycosylation of key
hemostatic proteins
One of the central functions of proteins from platelets, plasma, and
endothelial cells is the maintenance of vascular integrity accomplished through regulation of platelet activation, blood clotting,
fibrinolysis, and vascular repair. Together, these processes are
referred to as hemostasis. Key hemostatic proteins are major
therapeutic targets and, as such, site-specific O-glycosylation has
been thoroughly investigated on many of these proteins. We
434
KING et al
therefore compared O-glycosites identified on key hemostatic
proteins in this study with previously reported sites to (1) highlight
novel findings, (2) provide an updated summary of known
O-glycosites for the field, and (3) illustrate overall patterns of
O-glycosylation within protein families and across multiple protein
classes (Figures 3 and 4; supplemental Figure 6).50,51 For many sites,
this is the first evidence of native (in vivo) O-glycosylation of these
proteins (supplemental Figure 6). Examples of this can be seen
on Protein S, prothrombin, urokinase, carboxypeptidase B2, and
plasminogen activator inhibitor-1, among others (Figure 3; supplemental Figure 6). Similarly, although the platelet receptor glycoprotein
(GP)Ib-IX-V complex is known to be O-glycosylated, only a single
probable O-glycosite (Thr308 on GP1BA) has been described.51
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
A
B
Total glycoproteome
Hemostatic factor glycosylation
cell lines
plasma
platelet
(11
8)
plas
ma
(66
)
huv
ec (
51)
huvec
40
plat
10
elet
sites per protein:
C
ovc
ar3
(33
)
mda
231
(33
)
hep
g2 (
155
)
ags
(91
)
mcf
7 (3
0
)
hek
293
(26
)
hela
(18
)
mkn
45 (
48)
imr3
2 (2
6)
t47d
(9)
Unambiguous glycosites
Unambiguous glycosites
hemostatic
shading = protein name
Fibronectin O-glycosites
t47d (3)
imr32 (2)
mkn45 (9)
hela (12)
hek293 (12)
mcf7 (15)
mda231 (15)
ovcar3 (18)
46
278
279
280
281
283
287
294
402
410
448
449
604
617
658
667
935
1122
1198
1199
1200
1276
1282
1481
1546
1557
1565
1566
1567
1570
1575
1585
1627
1630
1641
1717
1791
1823
1826
1828
1829
1831
1833
1877
1914
1915
1922
1931
1999
2016
2020
2045
2060
2061
2064
2065
2067
2076
2153
2155
2158
2160
2163
2294
2341
2345
2346
2349
2354
2363
Sample
platelet (10)
plasma (6)
huvec (29)
hepg2 (52)
O-glycosites (a.a. position)
n-term
c-term
D
Cell-type dependent O-glycosylation
XYLT2
Identical
CTGF
huvec
MGT4B
Conserved
platelet
FAM3C
A4
TPST2
Differential
NID2
TGON2
Figure 5. Cells express unique O-glycoproteomes. To visualize the O-glycoproteome of each cell, the presence or absence of individual glycosites was illustrated using unclustered
heat maps. Glycosites were ordered alphabetically by Uniprot accession and subsequently by position in the protein. (A) Total O-glycosylation illustrating the overlap of unique, unambiguous
glycosites identified in each sample. (B) Comparison of O-glycosite identification on key hemostatic factors between different sample types. (C) A detailed view of differential O-glycosylation of
fibronectin across samples. Individual O-glycosites identified in fibronectin are plotted by sample type in order of position on the protein, with N-terminal sites plotted closest to the origin.
Numbers indicate total glycosite count per protein. (D) Differential O-glycosylation of proteins in the hemostatic system. The hemostatic O-glycoproteome was filtered to identify proteins
represented by .5 spectra in each sample and with similar spectral counts between samples (total protein spectral counts differing by ,10) to ensure comparable O-glycosite coverage was
achieved between samples. These proteins demonstrated both overlapping and also cell-specific glycosylation as exemplified in (D). Drawing is to scale; the arrowhead indicates the protein
extends beyond illustration. Shading indicates number of sites per protein (A), protein identity (B), or sample type (C). a.a., amino acid.
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
O-GLYCOSYLATION IN THE HEMOSTATIC SYSTEM
435
Here we were able to identify O-glycosites on all members of the
complex (Figure 4). Novel O-glycosites were also discovered on
integrin receptors, with the occurrence of O-glycosites within the
VWFA domain of b-integrins being of particular note. The most
conspicuous finding, however, was the extensive glycosylation of
coagulation factor V (FV), fibrinogen a, and fibronectin, which far
exceeded prior expectations.
O-glycosylation occurs in a cell-specific manner in the
hemostatic system
We next asked if proteins from different cellular sources were
differentially O-glycosylated. Cells and tissues express distinct
repertoires of GalNAc transferases (supplemental Figure 7) that
can potentially fine-tune protein function in a cell-specific
manner.52,53 Comparing the total O-glycoproteomes of each
cellular source revealed that platelets, endothelial cells, and
plasma proteins each express a unique O-glycoproteome
(Figure 5A). This cell-specific behavior was found to be more
pronounced when compared across a broader selection of cell
types (Figure 5B). Such differences could, however, result from
differential protein expression or GalNAc-T activity. Therefore, we
analyzed the cell-specific glycosylation of fibronectin, which has
been identified in multiple cell types (Figure 5C). We found
2 regions of glycosylation to be conserved (amino acid positions
278-287 and 2060-2067), but also observed multiple regions of
cell-specific glycosylation. To ensure similar coverage between
samples, we next analyzed the cell-specific glycosylation of
proteins with $5 spectra in each sample and with congruent
spectral counts. Again we found evidence for sample-specific
glycosylation in parallel with conserved glycosylation patterns
(Figure 5D). These findings open the possibility of cell-specific
regulation of hemostatic proteins by cell-specific differential
O-glycosylation.
An unbiased screen to identify protein features
colocalizing with O-glycosylation
The relative location of a PTM can be highly informative of its
potential function. For example, acetylation at a protein N-terminal
will likely alter the protein’s stability, whereas the same modification
on an active-site lysine may modify catalytic activity. We noted
during our manual analysis that glycosites were repeatedly found
near certain protein annotations such as phosphorylation sites,
proteolytic cleavage sites, serpin reactive center loops, and in helix/
coiled regions, VWF domains, and protein stems/linkers. These
subpatterns could suggest discrete roles for O-glycans worthy of
further investigation; however, such manual analysis is biased by
the types of proteins studied, prior knowledge, and potential effects
of nonrandom Ser/Thr distribution. Therefore, we performed a
systematic and unbiased association study using the full complement of protein annotations available from UniprotKB54 and all
O-glycosites described to date1,8,28-30,32,47,55-57 (supplemental
Table 1). This analysis identifies whether O-glycosites are over- or
underenriched around (6 15 amino acids) each protein annotation
relative to the expected frequency based on the background
distribution of Ser/Thr residues, and is somewhat analogous to
a gene ontology study, in which gene ontologies are replaced
with protein annotations (Figure 6A). Among the most significant
findings, individual O-glycosites were associated with cleavage
sites in accordance with previous data on proprotein convertase
436
KING et al
and metalloprotease cleavage.58,59 We also observed association
with propeptides and bioactive peptides, and further inspection
indicated that glycosites were localized within the processed region,
rather than around the periphery. Although rare, O-glycosites were
associated with a small group of protein domains including the
low-density lipoprotein receptor class A, thioredoxin, EF-hand,
Ig-like, and sushi domains. Signal and transmembrane domains
were under-represented as expected. Interestingly, N-glycosites,
nucleic acid binding regions, and binding regions in general were
less frequently associated with an O-glycosite than expected.
Although the data were supported by few sites, it was notable that
glycosites were also found in proximity to regions involved in
endoplasmic reticulum/Golgi trafficking and sulfotyrosine annotations. Identified subtypes of O-glycosylation including selected
examples are shown in Figure 6B.
O-glycosylation regulates cleavage of
hemostatic factors
From our analysis, it was apparent that a number of O-glycosites
were closely juxtaposed with cleavage sites critical to the regulation
of hemostasis. Therefore, we investigated whether O-glycans could
regulate hemostatic factor processing in vitro using synthetic
20-mer peptides. We first tested whether identified glycosites could
be glycosylated in vitro using the 3 major glycosyltransferases:
GalNAc-T1, T2, and T3. We found that 31 of the 33 peptides
tested could be glycosylated by $1 enzyme in overnight reactions,
with a substantial overlap in enzyme specificity (supplemental
Figure 8). We then selected 8 of the glycosylated peptides, which
also contained a reported cleavage site, and investigated whether
the presence of O-glycans altered the cleavage response in an in
vitro time-course assay (Figure 7). We found that O-glycosylation
partially inhibited the cleavage response to neutrophil elastase,
thrombin, and MMP12 in a glycosite- and glycoform-specific
manner, with a marked inhibition in the cleavage of proteaseactivated receptor 2 (PAR2) and tissue factor pathway inhibitor 1
(TFPI1) by neutrophil elastase and the cleavage of protein S by
thrombin. These findings indicate that O-glycosylation is able to
alter the sensitivity toward proteolytic cleavage across multiple
protein- and protease-families in the hemostatic system.
Discussion
Here we took advantage of recent progress in lectin enrichment and
high-resolution MS to selectively identify site-specific O-GalNAc
type glycosylation in platelets, plasma, and endothelial cells. With
this approach, we were able to substantially increase our knowledge
of the native human O-glycoproteome by unambiguously identifying
1123 sites on 649 proteins, including identification of 541 sites and
231 O-glycoproteins that had not previously been found in native
samples.30,57,60 It should be noted, however, that these data likely
underestimate the total number of O-glycans present because of the
inherent limitations of shotgun proteomics.27 This is of particular
concern in regards to mucin-like repeat regions and highly homologous
protein families that are refractory to mapping by mass spectrometry. In such cases, the NetOGlyc 4.0 O-glycosite predictor can
be used to complement experimental approaches, because
probable mucin-like regions can be identified by regions of
predicted high-density glycosylation, as exemplified by GP1BA42,61
and PSGL1.43,62
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
A
Protein Feature
286
Fold change
6
4
13 55
15
54 13
140 17 15 11
2
12
19 13 32
153 43 71 16
263 39 106 14 14 32
27 28
16 62
O
−G
Ta Th alN
nd ior Ac
em ed
Tr
o
af
Th Re xin
fic
yr pe
kin
og at
g
lo s
−
ER C bul
Pe a le in
a
LD ptid nd vag
L− e/ /or e
re Pro go
ce p lg
pt ep i
or tid
cl e
a
EF ss
Su − A
l h
O foty and
−
lin ros
C
ar
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bo
d
hy
(X
y
dr
at S l)
Pr
e− us
o/
Se
bi hi
nd
ra
nd Cy ing
s
/
Ig or −ri
−l Th ch
ike r−
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2− h
ty
I
pe
g
N −
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ke V il
d −t
(G yp
lc e
N
Si B Ac
gn in )
al din
p
Ac ep g
et tid
yll e
ys
EG
in
e
F/
EG LR
R
In F−l
te ike
ra
ct
io
n
M
Tr Ac et
an ti al
sm ve
em site
br
an
e
0
B
Cleavage site
Stem
GPIBA, GPV, GPVI
LRP8, PSGL1, Semaphorin 4D
rpt rpt rpt
Within domain
PAR2
FV, FVIII, FIX,
Thrombin,
Fibrinogen,
Kininogen
Linker
Mucin-type
rpt
APP
Plexin
Syndecan
MUC1, MUC5B
FV, FXII, GPIBA,
PlexinB1
thioredoxin
thyroglobulin
EF-hand
Sushi
TFPI, VWF
Thrombin
Plasminogen
Processed region
ADAM proteases
FV, FIX, FX
PDGF, Endothelin
P-selectin, complement
PDIAs, QSOX2, IGFBPs, nucleobindin
active protein
GalNAc monosaccharide
Figure 6. Enrichment of protein annotations around O-glycosites. (A) Protein annotations from the UniprotKB database were used to determine whether specific
protein features were enriched around (6 15 amino acids) O-glycosites relative to the background Ser/Thr distribution. Annotated features with a .1.2 fold-change and P , .01
(Fisher’s exact test, Bonferroni correction) are illustrated on the graph above. Note that O-glycosites often occurred in the vicinity of Ser/Thr phospho-sites and disulfide-bonded
Cys, but at the expected frequency. Numbers above the bars indicate the count of O-glycosites found in the vicinity of the annotation. Fold-change indicates the under- or
overenrichment of O-glycosites relative to the background frequency of Ser/Thr residues. Only annotations represented by .5 proteins and 10 glycosites were included in the
analysis. (B) Graphic depiction showing examples of the different types of O-glycosylation identified in this study. rpt, repeat.
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
O-GLYCOSYLATION IN THE HEMOSTATIC SYSTEM
437
Figure 7. Glycosylation inhibits proteolytic processing. In vitro cleavage
A
analysis of glycopeptides and their nonglycosylated counterparts. Synthetic
MMP12 MMP7
peptides were in vitro–glycosylated and then subjected to a protease digestion
time course with the indicated enzymes. (A) Summary of results. Identified
MMP12
NE
cleavage sites are indicated by an arrow. Proteases affected by glycosylation are
colored blue (partial inhibition) or red (complete inhibition). Glycosites identified
by prior glycoproteomic analysis are shown in red; glycosites identified by
MMP12
MMP12
LC-MS/MS on in vitro glycosylated peptides are indicated by a yellow box.
Glycosylation of the TFPI1_2 peptide both delayed and repositioned NE
THRB
*
cleavage. (B) Example matrix-assisted laser desorption/ionization–time-of-flight
spectra showing glycoform-dependent inhibition of thrombin serine protease
THRB
*
*
alternate glycoforms
activity. The monoisotopic mass of the cleavage product is indicated in red. NE,
neutrophil elastase; THRB, thrombin; PLMN, Plasmin. n 5 2.
GalNAc
monosaccharide
THRB
PLMN
*
FXa
FXa
*
THRB
THRB
NE
The identification of a large number of O-glycosites enabled
investigation of the potential function of glycosylation by determining which annotated protein features colocalize with
O-glycosites. Here, we assessed whether glycosites were over- or
underenriched around protein features compared with the background distribution of Ser/Thr residues. As expected, we found
glycosites to be enriched around tandem repeat or Pro/Ser/Thr rich
regions, presumably indicative of canonical mucin-type glycosylation.
Within the hemostatic system, the coagulation factors V and XII,
GP1BA, PSGL1, and Plexin B1 were identified with glycosites in such
regions. This form of dense mucin-type glycosylation is thought to
confer lubricating properties,63 extend the polypeptide backbone,
reduce flexibility, and provide resistance to proteases.7,64 For
example, O-glycans are thought to extend GP1BA from the platelet
surface, allowing optimal interaction with VWF under shear stress.21
The presence of a mucin-type domain in the soluble coagulation
factors could conceivably protect the factors from degradation and
premature activation in circulation, but such a role has not yet
been described. We also found a strong association between
O-glycosylation and proteolytic cleavage sites. Accumulating data
suggest that this colocalization is not simply the result of a propensity
for such PTMs to occur in similar regions, because loss of
O-glycosites has been directly shown to inhibit or activate protein
processing by proteases.58,59,65 In the context of the hemostatic
system, sialylation and O-glycosylation of VWF alter sensitivity to
cleavage in a protease-specific manner.66 Similarly, O-glycosylation
regulates the rate of cleavage of coagulation factor X22 and LRP867;
and loss of extended O-glycans results in degradation of platelet
GP1BA in Cosmc null mice.25 In the present study, glycosites were
found juxtaposed with critical processing sites in a large number of
key hemostatic proteins (Figure 5B). Furthermore, these data were
supported by in vitro assays demonstrating that cleavage of Protein S
by thrombin and cleavage of PAR2 and TFPI1 by neutrophil elastase
438
KING et al
are inhibited by the presence of O-glycosylation. Such results suggest
that O-glycosylation might be a major regulator of protein stability and
processing in hemostasis.
In addition to mucin-type regions and proteolysis, we found
O-glycosites within propeptides and bioactive-peptides, which are
proteolytically removed from the mature protein during hemostasis.
Glycosylation within these regions was common on hemostatic
factors, particularly coagulation factors and collagens. The functional
consequences of this type of O-glycosylation have not been
elucidated yet; however, because the majority of O-glycans carry
at least one negatively charged sialic acid, the release of these
regions facilitates a rapid change in the charge density of the
protein and thereby may have substantial effect on protein
confirmation and binding. Additional associations with tyrosine
sulfation and endoplasmic reticulum/Golgi trafficking were noted.
Tyrosine sulfation contributes to protein-protein binding68 and is of
interest because coordinated binding of a Core 2 O-glycan and
sulfated tyrosines on the N-terminal of PSGL1 is critical to its
recognition by P-selectin.19,20 Glycosites were found in proximity
to sulfotyrosines in coagulation factors VIII, IX, and XII, and also
vitronectin, GP1BA, and nidogen 1. Similarly, proximity to endoplasmic reticulum/Golgi trafficking signals is notable because
O-glycosylation has been demonstrated to regulate secretion,
suggesting a function in vesicular transport.69-71 Given that vesicular
trafficking signals remain poorly annotated, it will be of interest to
further investigate the potential role of O-glycosylation in protein
trafficking experimentally.
The function of O-glycans is not solely dependent on the position
of the glycan, but also, to a large extent, elongation and the addition
of terminal structures. Such structures act as ligands for
carbohydrate-binding proteins and are often altered in pathogenic
states (eg, in response to inflammatory signaling72 and during
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
naked peptide
x105
3
2
8000
2
2
200
100
0
x104
6
1
2
0
x105
1.5
4
0.5
0
0.0
2500
2279.062
1.0
2
2000
1505.67
4
0
x104
6
0
0
300
3000
2482.160
1505.667
1000
1500
2000
2500
0 hr
2482.482
6000
4000
2000
3 hr
0
x104
2.0
1.5
1.0
0.5
0.0
x105
1.25
1.00
0.75
0.50
0.25
0.00
Intens. [a.u.]
Intens. [a.u.]
1302.672
2482.48
4
Intens. [a.u.]
Intens. [a.u.]
2076.019
1500
x104
1
1302.67
1000
2279.06
Intens. [a.u.]
2076.02
Intens. [a.u.]
Intens. [a.u.]
x105
5
4
3
2
1
0
5000
4000
3000
2000
1000
0
x104
3
+ 50nM
thrombin
T3 glycoform
T1 glycoform
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
B
3000
12 hr
22 hr
1000
1500
2000
2500
3000
Thrombin peptide 187-206 (THRB_1)
0.5
1881.099
Intens. [a.u.]
Intens. [a.u.]
0.0
x104
4
3
2
1
0
x104
4
3
2
1
0
x104
0.8
0.6
0.4
0.2
0.0
1000
1500
2000
2500
0.0
x104
1.00
0.75
0.50
0.25
0.00
x104
2.5
2.0
1.5
1.0
0.5
0.0
x104
Intens. [a.u.]
2431.375
2084.25
2084.253
Intens. [a.u.]
1881.10
0.5
Intens. [a.u.]
2227.605
2431.38
1.0
Intens. [a.u.]
0.0
x104
1.0
Intens. [a.u.]
0.5
2
1
0
x104
1.00
0.75
0.50
0.25
0.00
Intens. [a.u.]
Intens. [a.u.]
2227.61
1.0
x104
1000
1500
2000
+ 50nM
thrombin
T3 glycoform
T1 glycoform
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
Intens. [a.u.]
naked peptide
x105
2500
x104
2
2635.89
1
0 min
0
x104
3
2
1
0
4
x104
10 min
3
2
1
0
x104
1.5
1.0
0.5
0.0
2287.78
30 min
60 min
6000
4000
2000
3 hr
0
1000
1500
2000
2500
Protein S peptide 95-114 (PROS1)
Figure 7. (Continued).
metastasis73). It is not currently possible to simultaneously identify
both glycan structure and glycosite for complex O-glycans.
A number of glycopeptides identified in this study, however, were
predicted to carry GlcNAc residues suggestive of elongated/
branched structures. We were able to predict the presence of such
branched structures on GP1BA at 490-494 in keeping with
previous reports indicating that the majority of GP1BA O-glycans
were of Core 2 structure.74,75 In addition to GP1BA, a number of
key hemostatic factors, including GP1BB, GPIX, GPVI, fibronectin,
coagulation factors V and XII, and P-selectin, carried branched
structures suggesting that complex O-glycans (including blood
group ligands) may be present on a substantial number of
extracellular proteins. Notably, most sites identified carrying a
branched structure were also identified with a simple Core 1
structure, indicating that there is variability in both site occupancy
and glycan structure at individual sites.
In summary, O-glycosylation is a widespread modification of
platelet, plasma, and endothelial cell proteins, occurring in
disordered regions of extracellular proteins and presenting with
variable occupancy and composition at individual sites. Although
28 FEBRUARY 2017 x VOLUME 1, NUMBER 7
the precise role of O-glycans in hemostasis remains enigmatic,
the distinct patterns of O-glycosylation identified here suggest the
presence of multiple autonomous subgroups and provide a roadmap
for future structure-function studies. Such information will be critical
in the production of biotherapeutics and development of glycanbased biomarker studies.
Acknowledgments
The authors thank Claus Ekstrom for statistical advice provided during
the writing of this manuscript, Annika Lindkvist for assistance in the
culture of HUVECs, and Simon Kuijpers for assistance with illustration.
This work was supported by The Danish Research Councils
(Sapere Aude Research Leader grant [H.W.] and Talent Grant
[K.T.S.]), The Mizutani Foundation, Kirsten og Freddy Johansen
Fonden, A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til
Almene Formaal, The Novo Nordisk Foundation, the Danish
Strategic Research Council, the Lundbeck foundation, the program
of excellence from the University of Copenhagen (CDO2016),
and The Danish National Research Foundation (DNRF107).
O-GLYCOSYLATION IN THE HEMOSTATIC SYSTEM
439
Authorship
Contribution: S.L.K., H.J.J., K.T.S., S.Y.V., M.H.D., A.W., and H.H.W.
designed experiments and wrote the paper; S.L.K., K.T.S., and T.D.M.
performed experiments; and S.L.K., K.T.S., H.J.J., A.H., and S.Y.V.
analyzed data.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Hans H. Wandall, Department of Cellular and
Molecular Medicine, Centre for Glycomics, University of Copenhagen,
DK-2200 Copenhagen N, Denmark; e-mail: [email protected].
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