(Received for publication, September 4, 1996, and in revised form, November 22, 1996)
From the Human plasminogen, the inactive precursor of
plasmin, exists in two major glycoforms. Plasminogen 1 contains an
N-linked oligosaccharide at Asn-289 and an
O-linked oligosaccharide at Thr-345. Plasminogen 2 is known
to contain only an O-linked oligosaccharide at Thr-345. However, plasminogen 2 displays a further well documented
microheterogeneity dependent on the N-acetylneuraminic acid
content, which has functional consequences with regard to activation of
plasminogen. The proposed structure and number of known oligosaccharide
linkages in plasminogen 2 is insufficient to account for this
microheterogeneity. In the present study, a combination of trypsin
digestion, lectin affinity chromatography, Edman degradation amino acid
sequence analysis, carbohydrate composition analysis, and mass
spectrometry revealed the existence of a novel site for
O-linked glycosylation on plasminogen 2 at Ser-248.
Direct evidence for the structure of the carbohydrate was
obtained from a combination of lectin affinity chromatography, desialylation experiments, and mass spectrometry analysis. These findings provide a structural basis for some of the observed
microheterogeneity, and have implications with regard to the known
functional consequences of the extent of sialylation of
plasminogen.
Plasminogen (Pg)1 is the inactive
precursor of plasmin, quantitatively the most important proteinase
involved in fibrinolysis. Pg exists in two major glycoforms, Pg 1, which possesses an N-linked high mannose-type carbohydrate
chain located at Asp-289 and an O-linked carbohydrate chain
linked at Thr-345, and Pg 2, which contains only the carbohydrate chain
present at Thr-345 (1, 2). The sialylated O-linked
carbohydrate chain at Thr-345 contains N-acetylneuraminic
acid (NeuNAc), galactose (Gal), and N-acetylgalactosamine (GalNAc) and has the structure NeuNAc It has long been known that Pg 2 can be resolved by isoelectric
focusing techniques into at least six glycoforms that differ only in
their N-acetylneuraminic acid (NeuNAc) content (4, 5)
Recently, we have separated the glycoforms of human Pg 2 by employing a
combination of lectin affinity chromatography and chromatofocusing (6).
Our data showed that the NeuNAc content of Pg 2 glycoforms varied from
1.3 mols/mol of protein to 13.65 mols/mol (6). Furthermore, the
individual Pg 2 glycoforms display markedly different kinetic behavior
when activated with tissue-type plasminogen activator (tPA),
urinary-type plasminogen activator (uPA), and streptokinase (6, 7).
Activation of Pg 2 by tPA was most dependent on NeuNAc content with a
steady decrease in catalytic efficiency with increased sialylation,
whereas catalytic efficiencies of activation by uPA appeared to be
unaffected up to a threshold of NeuNAc content. The most highly
sialylated glycoform of Pg 2 was essentially resistant to activation by
both tPA and uPA (7). Interestingly, streptokinase activation of human
Pg was also regulated by NeuNAc content, with the most highly
sialylated glycoform activated 20-fold less efficiently than the least
sialylated glycoform. Although carbohydrate did not stop streptokinase
forming an initial activator complex with human Pg, as demonstrated by gel filtration experiments, the carbohydrate was hypothesized to
interfere with the stability of the Michaelis complex (7). In contrast
to the effect on Pg activation, NeuNAc content did not interfere with
the inhibition of generated plasmin glycoforms by
Further evidence that the glycosylation of Pg modulates the functional
activity of the protein has been provided by Mori et al.
(8), who demonstrated differential activation of Pg 1 and 2 by tPA I
and II, respectively. Davidson and Castellino (9) have shown that
differently glycosylated forms of Pg exhibit different kinetic
parameters for activation by uPA. In addition, neonatal Pg 2, which has
18 times more NeuNAc than adult Pg 2, is activated 6-fold less
efficiently by tPA (10). Pg 2 also binds to cell surfaces with greater
affinity than the more glycosylated Pg 1 (11). Unglycosylated Pg,
expressed in Escherichia coli, was resistant to activation
by tPA and uPA and was cleared significantly faster than glycosylated
Pg molecules (5). Together, these data suggest an important role for
carbohydrate, in general, and NeuNAc, in particular, in regulating the
function of Pg.
Examination of the proposed structure of the carbohydrate chain on
Thr-345 of Pg 2 (3) predicts the possibility of only two glycoforms
based on the NeuNAc content; however, the existence of at least six
glycoforms is well documented (5, 6). To address this apparent
discrepancy, we have reassessed the glycosylation of Pg2.
In this study, we present data derived from amino-terminal sequence
analysis of tryptic peptides, mass spectrometry, and
fluorophore-assisted carbohydrate analysis (FACE), demonstrating that
Pg 2 contains a novel O-linked carbohydrate chain linked to
Ser-248.
Pg 2 was purified from fresh frozen plasma
(American Red Cross, Durham, NC) as described previously using a
combination of lysine-Sepharose and concanavalin A-Sepharose affinity
chromatography (6). Each batch of Pg 2 was purified from 4-8 units of
fresh frozen plasma. At least three separate batches of prepared Pg 2 were used in these studies. Trypsin was obtained from
Sigma. Jacalin-agarose (4 mg/ml) was obtained from
Vector Labs Inc. (Burlingame, CA). Neuraminidase (NANase III) was a
kind gift from GLYKO (Novato, CA). Proteinase SV8 was obtained from
Boehringer Mannheim.
Dithiothreitol, iodoacetamide,
Peptides were produced by limited proteolysis
essentially as described previously (12). Briefly, Pg 2 (2-5 mg/ml)
was incubated for 2 h at 37 °C in 6 M guanidine
hydrochloride, 0.1 M Tris-HCl, pH 8.2, 10 mM
dithiothreitol. The solution was adjusted to 30 mM
iodoacetamide and incubated in the dark at room temperature for 30 min.
After dialysis against 0.1 M Tris-HCl, pH 8.2, overnight, the Pg 2 solution (including precipitated material) was transferred to
a 50-ml conical tube, and trypsin was added such that the final molar
ratio of Pg to trypsin was 100:1. The reaction was allowed to proceed
for 4-16 h at 37 °C, after which the solution was adjusted to pH
7.0 with HCl. Peptides containing an O-linked carbohydrate chain were purified by applying the peptide mixture to a
jacalin-agarose column (16 × 100 mm) equilibrated in 100 mM Tris-HCl, pH 7.0. Jacalin is a lectin with affinity for
the disaccharide
1- Automated Edman
degradation was performed on an Applied Biosystems 477A pulsed liquid
phase sequencer with on-line phenylthiohydantoin analysis using an
Applied Biosystems 120A HPLC system operated according to manufacturer
recommendations. Peptide concentrations were determined using amino
acid composition analysis. Peptide samples (approximately 500 pmol)
were hydrolyzed for 24 h at 110 °C in 6 N HCl
containing 0.1% phenol (14). The tubes were evacuated and flushed with
nitrogen several times before they were sealed under vacuum. The
hydrolysates were analyzed in a Beckman 6300 amino acid analyzer with
an on-line Hewlett-Packard 3390A integrator and using sodium citrate
buffers provided by the manufacturer.
The
monosaccharide composition of the peptides was determined using FACE
technology (Glyko, Novato, CA). Briefly, 200 pmol of each peptide
(determined by amino acid analysis) were subjected to three separate
hydrolysis reactions to determine the presence of NeuNAc (0.2 N TFA, 80 °C × 1 h), amine sugars (8 N TFA, 100 °C × 3 h), and neutral sugars (4 N TFA, 100 °C × 5 h). The released monosaccharides were then labeled overnight at 37 °C with a
fluorescent label as described by the manufacturer, and the labeled
monosaccharides were resolved on a proprietary gel system. The gels
were imaged using a fluorescence camera linked to a computer, and data
were analyzed using proprietary software.
Measurements were made on a Fisons VG Quattro-BQ triple
quadrupole mass spectrometer equipped with a pneumatically assisted electrostatic ion source operating at atmospheric pressure and controlled using the MassLynxTM data system (Version 2.0).
The glycopeptides, isolated as described above, were lyophilized and
resuspended in aqueous acetonitrile (50%) containing 1% formic acid.
Spectra were acquired in the multi-channel acquisition mode from
mass/charge (m/z) 600-1600 with a scan time of
10 s. For some experiments, the reversed phase fractions were
resubjected to reverse phase-HPLC (Deltabond, ODS 150 × 1 mm,
Keystone Scientific, Bellefonte, PA) using an Isco (Lincoln, NA)
microbore system. The effluent was monitored at an absorbance of 216 nm
and split evenly into two streams. One stream was fed directly to the
ion source of the mass spectrometer. Spectra were acquired in continuum
mode from mass/charge (m/z) ratio of 600-1600
with a scan time of 5 s. The mass scale was calibrated with horse
heart myoglobin (Mr 16951.48) and with a resolution corresponding to a peak width at half-height of 1.0 Da for
m/z 893. The mass spectra were transformed to a
molecular mass using software supplied by the manufacturer.
The glycopeptides purified
by jacalin-agarose affinity chromatography resolved into three
peptides, eluting at 20, 27, and 27.5% acetonitrile (Fig.
1). Although the peptides eluting at 27 (pep2) and 27.5% (pep3) acetonitrile have been
previously described (12), the peptide eluting at 20%
(pep1) acetonitrile is novel. Edman degradation amino acid
sequence analysis of the first 20 amino acid residues of peptides 2 and
3 confirmed that both of these peptides were derived from Pg 2 and
consisted of a fragment commencing at Ile-329 (Table I).
Confirmation of the sequence of the last 18 residues was obtained by
further digesting peptides 2 and 3 with SV8 as described under
"Experimental Procedures." The identity of pep1 was obtained by
amino acid sequencing of the entire tryptic peptide. The sequences
(Table I) differed slightly from the Pg sequence previously published.
No phenylthiohydantoin amino acid derivative was detected in the cycle
during Edman degradation at position Thr-345 in peptides 2 and 3, consistent with a modification of the threonine by the known
O-linked carbohydrate chain present at this residue (3).
Ser-338 in peptide 2 was also modified and both peptides 2 and 3 had
Gln-341 instead of the expected Glu-341, as reported previously (12).
Peptides 2 and 3 always eluted from the C18 column in equimolar
concentrations as indicated by peak area (data not shown).
Primary structure of glycopeptides pep1, pep2 and pep3
Department of Pathology,
Department of Pediatrics,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
2-3Gal-
1-3GalNAc. In
1-5% of Pg molecules, there is a further NeuNAc linked directly to the GalNAc (1, 3).
2-antiplasmin (6).
Proteins
-methylgalactopyranoside, and all buffer chemicals were purchased
from Sigma. All other reagents were of reagent grade
quality.
-galactopyranosyl-3-(
-2-acetamido-2-deoxygalactopyranoside), the core disaccharide of mucin-type carbohydrate chains (13). The
column was washed in 100 mM Tris-HCl buffer (3 column
volumes), and bound peptides were eluted using 10 ml of 100 mM Tris-HCl, 20 mM
-methylgalactopyranoside.
Glycopeptides were further purified using HPLC. Separations were
performed on an octadecylsilane (C18) column (4.6 × 250 mm, 5 µm particle size) using a linear gradient (0.5% min
1)
of 0.1% trifluoroacetic acid (TFA) in acetonitrile at a flow rate of
0.5 ml/min
1. Elution of peptides was monitored at an
absorbance of 214 nm. Peptides obtained were sequenced as described
below. Some peptides were further digested using SV8 proteinase.
Briefly, glycopeptides (obtained as described above) were lyophilized,
and dissolved in 100 mM Tris-HCl, pH 8.0. The peptides were
then incubated with SV8 (1:100 molar ratio of proteinase:peptide) at
37 °C overnight. Peptides were then separated on C18 HPLC, using a
0-40% acetonitrile gradient and sequenced.
HPLC Separation of Glycopeptides
Fig. 1.
Chromatograph of HPLC of jacalin-agarose
affinity purified tryptic peptides of Pg 2. The chromatograph
shows the elution profile of a C18 column. Peptides were resolved using
a 0-40% acetonitrile gradient. All buffers were 0.1% TFA. The flow
rate was 0.5 ml min1. Peptides were monitored by
measuring the absorbance at a wavelength of 214 nm.
[View Larger Version of this Image (18K GIF file)]
Peptide
Sequence
Peptide
1
242CTTPPPXSGPTYQCLK-257
Peptide
2
329IPSCDSSPVXTEQLAPXAPPELTPVVQDCYHGDGQSYR
366
Peptide
3
329IPSCDSSPVSTEQLAPXAPPELTPVVQDCYHGDGQSYR
366
Amino acid sequence analysis of peptide 1 (Table I) indicated that it was a Pg 2 derived fragment, commencing at Cys-242 and terminating at Lys-257. There was a blank cycle at position Ser-248 in this peptide tide, indicating a modification of the serine. The amount of peptide 1 obtained from HPLC was consistently 13-15% of the amounts of peptides 2 or 3, suggesting that not all Pg 2 molecules have this modification.
Mass Spectrometry AnalysisTo determine the nature of the modification inferred by the blank cycles found during sequence analysis, we performed ES-MS analysis. ES-MS analysis of peptide 1 (Table II) revealed a mass of 2449.9 Da. The expected mass of the unmodified carboxyamidomethylated peptide is 1796.0 Da. As the predicted mass of a mucin carbohydrate trisaccharide chain attached to the side chain of serine or threonine is 656.6 Da, this indicates that Ser-248 may possess an O-linked mucin carbohydrate chain.
|
Similar analysis of peptide 2 revealed a mass of 4896.6 ± 1 Da (Table II). The expected mass of the carboxyamidomethylated peptide is 4162.5. Mass spectrometry analysis of peptide 3 consistently revealed a peptide with mass value of 4816.2 ± 0.6 Da (Table II). Thus, peptides 1, 2, and 3 all have a greater mass than would be expected from a simple analysis of the primary sequence, indicating that all peptides isolated from jacalin-agarose are modified by the addition of at least one trisaccharide moiety on Ser or Thr. To confirm the composition and structure of the carbohydrate on these glycopeptides we performed FACE analysis.
FACE AnalysisPeptides 1, 2, and 3 were analyzed by FACE to
determine carbohydrate composition. A representative analysis is shown
in Fig. 2. The only monosaccharides detected in peptides
1, 2, and 3 were galactose, N-acetylgalactosamine, and
NeuNAc. The ratios of N-acetylgalactosamine to protein
(Table III) are essentially equimolar for peptides 2 and
3, indicating that the additional modification of Ser-338 noted in
peptide 2 is unlikely to be a carbohydrate modification.
|
Glycopeptides 1-3 were
treated with NANase III, a neuraminidase with specificity for
(2-3),
(2-6), and
(2-8) linkages for 16 h at 37 °C.
The peptides were then analyzed by ES-MS. The mass differences before
and after NANase III treatment are consistent with the loss of the
NeuNAc (Table IV), indicating that each of the
glycopeptides purified only has one NeuNAc residue.
|
In this study, we provide evidence that Pg 2 molecules contain an
additional O-linked carbohydrate chain and have localized the site of attachment of this second carbohydrate chain to Ser-248. We
have isolated three jacalin-reactive peptides from Pg 2, of which one
(peptide 1) is a novel glycopeptide. The other two peptides, designated
peptides 2 and 3, have been described previously (12). The mass
spectrometry and carbohydrate composition analysis of jacalin-purified
peptide 1 indicates that the carbohydrate attached to Ser-248 of Pg 2 has the structure NeuNAc2-3Gal
1-3GalNAc, identical to the known
structure of the carbohydrate chain on Thr-345 of Pg 2.
Pg contains five kringles that mediate binding to substrate surfaces, such as fibrin and fibronectin and cell receptors, and that regulate the activation of Pg as well as plasmin activity. The site of attachment for the novel sialylated trisaccharide chain described here is located between kringles 2 and 3. We and others (6, 8) have demonstrated the importance of carbohydrate in the regulation of Pg activation by tPA. We have also previously demonstrated that a decrease in the catalytic efficiency of Pg activation by tPA correlates with increasing NeuNAc content (6, 7). It has been suggested that activation of Pg by tPA may rely on a very precise alignment of substrate and activator (15). Thus, the presence of more than one sialylated trisaccharide chain between kringle domains on Pg 2 molecules may disrupt important protein-protein interactions, leading to the observed reduced activation efficiency and providing a structural basis for this functional phenomenon. Table V displays a summary of data derived from the literature demonstrating the correlation between NeuNAc content and decreasing activation efficiency by tPA.
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Since there are at least six glycoforms of Pg 2 (4, 5, 6), the presence of a second sialylated trisaccharide on Ser-248 does not fully account for all the known microheterogeneity of this protein. There are other types of O-linked saccharide chains that can contain NeuNAc, notably oligosaccharide chains attached via a fucose residue (16) that are found in a variety of proteins involved in coagulation (17). The presence of such carbohydrate chains on plasminogen or the possibility of polysialic acid (6) must also be considered. Our data also demonstrate a modification of Ser-338 of plasminogen 2. Hortin and Yu (18) have presented evidence indicating that plasminogen 2 is phosphorylated at Ser-338. The mass difference of 80 Da we report here between peptides 2 and 3 provides further evidence in support of this hypothesis.
In conclusion, we provide evidence for a novel O-linked carbohydrate chain on Pg 2. This chain, attached at Ser-248, is a trisaccharide terminated with NeuNAc. The presence of this trisaccharide between kringles 2 and 3, coupled with the previously reported trisaccharide between kringles 3 and 4 (3), provides a structural basis for the observed correlation of NeuNAc content with structural and functional microheterogeneity.
The authors thank Mario Gonzalez-Gronow for useful discussion, Zuzana Valnickova for performing the Edman degradation amino acid sequence analysis, and Ida B. Thøgersen for performing the amino acid analysis. We also thank Hanne Grøn, Tammy Moser, and David Morgan for critical reading of the manuscript. Special thanks go to Sharon Stack for invaluable help with the manuscript.