Evidence for a Novel O-Linked Sialylated Trisaccharide onSer-248 of Human Plasminogen 2*

(Received for publication, September 4, 1996, and in revised form, November 22, 1996)

Steven R. Pirie-Shepherd‡§¶, Robert D. Stevensi, Nancy L. Andon‡**, Jan J. Enghild‡,and Salvatore V. Pizzo‡ ‡‡

From the ‡Department of Pathology, Duke University Medical Center and the iDepartment of Pediatrics,Mass Spectrometry Facility, Duke University Medical Center, Durham, North Carolina 27710

Human plasminogen, the inactive precursor of plas-min, exists in two major glycoforms. Plasminogen 1 con-tains an N-linked oligosaccharide at Asn-289 and an O-linked oligosaccharide at Thr-345. Plasminogen 2 isknown to contain only an O-linked oligosaccharide atThr-345. However, plasminogen 2 displays a further welldocumented microheterogeneity dependent on the N-acetylneuraminic acid content, which has functionalconsequences with regard to activation of plasminogen.The proposed structure and number of known oligosac-charide linkages in plasminogen 2 is insufficient to ac-count for this microheterogeneity. In the present study,a combination of trypsin digestion, lectin affinity chro-matography, Edman degradation amino acid sequenceanalysis, carbohydrate composition analysis, and massspectrometry revealed the existence of a novel site forO-linked glycosylation on plasminogen 2 at Ser-248. Di-rect evidence for the structure of the carbohydratewas obtained from a combination of lectin affinitychromatography, desialylation experiments, and massspectrometry analysis. These findings provide a struc-tural basis for some of the observed microheterogene-ity, and have implications with regard to the knownfunctional consequences of the extent of sialylation ofplasminogen.

Plasminogen (Pg)1 is the inactive precursor of plasmin, quan-titatively the most important proteinase involved in fibrinoly-sis. Pg exists in two major glycoforms, Pg 1, which possesses anN-linked high mannose-type carbohydrate chain located atAsp-289 and anO-linked carbohydrate chain linked at Thr-345,

and Pg 2, which contains only the carbohydrate chain presentat Thr-345 (1, 2). The sialylatedO-linked carbohydrate chain atThr-345 containsN-acetylneuraminic acid (NeuNAc), galactose(Gal), and N-acetylgalactosamine (GalNAc) and has the struc-ture NeuNAca2–3Gal-b1–3GalNAc. In 1–5% of Pg molecules,there is a further NeuNAc linked directly to the GalNAc (1, 3).It has long been known that Pg 2 can be resolved by isoelec-

tric focusing techniques into at least six glycoforms that differonly in their N-acetylneuraminic acid (NeuNAc) content (4, 5)Recently, we have separated the glycoforms of human Pg 2 byemploying a combination of lectin affinity chromatography andchromatofocusing (6). Our data showed that the NeuNAc con-tent of Pg 2 glycoforms varied from 1.3 mols/mol of protein to13.65 mols/mol (6). Furthermore, the individual Pg 2 glyco-forms display markedly different kinetic behavior when acti-vated 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 NeuNAccontent with a steady decrease in catalytic efficiency with in-creased sialylation, whereas catalytic efficiencies of activationby uPA appeared to be unaffected up to a threshold of NeuNAccontent. The most highly sialylated glycoform of Pg 2 wasessentially resistant to activation by both tPA and uPA (7).Interestingly, streptokinase activation of human Pg was alsoregulated by NeuNAc content, with the most highly sialylatedglycoform activated 20-fold less efficiently than the least sialy-lated glycoform. Although carbohydrate did not stop streptoki-nase forming an initial activator complex with human Pg, asdemonstrated by gel filtration experiments, the carbohydratewas hypothesized to interfere with the stability of the Michae-lis complex (7). In contrast to the effect on Pg activation, Neu-NAc content did not interfere with the inhibition of generatedplasmin glycoforms by a2-antiplasmin (6).Further evidence that the glycosylation of Pg modulates the

functional activity of the protein has been provided by Mori etal. (8), who demonstrated differential activation of Pg 1 and 2by tPA I and II, respectively. Davidson and Castellino (9) haveshown that differently glycosylated forms of Pg exhibit differ-ent kinetic parameters for activation by uPA. In addition, neo-natal 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 tocell surfaces with greater affinity than the more glycosylatedPg 1 (11). Unglycosylated Pg, expressed in Escherichia coli, wasresistant to activation by tPA and uPA and was cleared signif-icantly faster than glycosylated Pg molecules (5). Together,these data suggest an important role for carbohydrate, in gen-eral, and NeuNAc, in particular, in regulating the function ofPg.Examination of the proposed structure of the carbohydrate

chain on Thr-345 of Pg 2 (3) predicts the possibility of only twoglycoforms based on the NeuNAc content; however, the exist-

* This work was supported by National Institutes of Health ResearchGrant HL-24066 and the Mizutani Glycoscience Foundation. Supportfor the purchase of the mass spectrometer was supplied by the NorthCarolina Biotechnology Center. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.§ Current address: Rosenstiel Basic Medical Science Research Cen-

ter, Brandeis University, Waltham, MA 02254.¶ To whom correspondence should be addressed: Rosenstiel Basic

Medical Research Center, Brandeis University, South St., Waltham,MA 02254. Tel.: 617-736-2468; Fax 617-736-2419; E-mail: pirie@auriga.rose.brandeis.edu.** Current address: Center for Cancer Research, M.I.T., Cambridge,

MA 02139.‡‡ To whom reprint requests should be addressed: Dept. of Pathology,

Box 3712, Duke University Medical Center, Durham, NC 27710.1 The abbreviations and trivial names used are: Pg, plasminogen(s);

NeuNAc, N-acetylneuraminic acid; GalNAc, N-acetylgalactosamine;NANase, neuraminidase; tPA, tissue-type plasminogen activator; uPA,urinary-type plasminogen activator; FACE, fluorophore-assisted car-bohydrate analysis; HPLC, high performance liquid chromatography;TFA, trifluoroacetic acid; ES-MS, electrospray ionization-massspectrometry.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 11, Issue of March 14, pp. 7408–7411, 1997© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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ence of at least six glycoforms is well documented (5, 6). Toaddress this apparent discrepancy, we have reassessed theglycosylation of Pg2.In this study, we present data derived from amino-terminal

sequence analysis of tryptic peptides, mass spectrometry, andfluorophore-assisted carbohydrate analysis (FACE), demon-strating that Pg 2 contains a novel O-linked carbohydratechain linked to Ser-248.

EXPERIMENTAL PROCEDURES

Proteins—Pg 2 was purified from fresh frozen plasma (American RedCross, Durham, NC) as described previously using a combination oflysine-Sepharose and concanavalin A-Sepharose affinity chromatogra-phy (6). Each batch of Pg 2 was purified from 4–8 units of fresh frozenplasma. At least three separate batches of prepared Pg 2 were used inthese studies. Trypsin was obtained from Sigma. Jacalin-agarose (4mg/ml) was obtained from Vector Labs Inc. (Burlingame, CA). Neura-minidase (NANase III) was a kind gift from GLYKO (Novato, CA).Proteinase SV8 was obtained from Boehringer Mannheim.Chemicals—Dithiothreitol, iodoacetamide, a-methylgalactopyrano-

side, and all buffer chemicals were purchased from Sigma. All otherreagents were of reagent grade quality.Peptides—Peptides were produced by limited proteolysis essentially

as described previously (12). Briefly, Pg 2 (2–5 mg/ml) was incubated for2 h at 37 °C in 6 M guanidine hydrochloride, 0.1 M Tris-HCl, pH 8.2, 10mM dithiothreitol. The solution was adjusted to 30 mM iodoacetamideand incubated in the dark at room temperature for 30 min. Afterdialysis against 0.1 M Tris-HCl, pH 8.2, overnight, the Pg 2 solution(including precipitated material) was transferred to a 50-ml conicaltube, and trypsin was added such that the final molar ratio of Pg totrypsin was 100:1. The reaction was allowed to proceed for 4–16 h at37 °C, after which the solution was adjusted to pH 7.0 with HCl.Peptides containing an O-linked carbohydrate chain were purified byapplying the peptide mixture to a jacalin-agarose column (16 3 100mm) equilibrated in 100 mM Tris-HCl, pH 7.0. Jacalin is a lectin withaffinity for the disaccharide 1-b-galactopyranosyl-3-(a-2-acetamido-2-deoxygalactopyranoside), the core disaccharide of mucin-type carbohy-drate chains (13). The column was washed in 100 mM Tris-HCl buffer (3column volumes), and bound peptides were eluted using 10 ml of 100mM Tris-HCl, 20 mM a-methylgalactopyranoside. Glycopeptides werefurther purified using HPLC. Separations were performed on an octa-decylsilane (C18) column (4.6 3 250 mm, 5 mm particle size) using alinear gradient (0.5% min21) of 0.1% trifluoroacetic acid (TFA) in ace-tonitrile at a flow rate of 0.5 ml/min21. Elution of peptides was moni-tored at an absorbance of 214 nm. Peptides obtained were sequenced asdescribed below. Some peptides were further digested using SV8 pro-teinase. Briefly, glycopeptides (obtained as described above) were ly-ophilized, and dissolved in 100 mM Tris-HCl, pH 8.0. The peptides werethen incubated with SV8 (1:100 molar ratio of proteinase:peptide) at37 °C overnight. Peptides were then separated on C18 HPLC, using a0–40% acetonitrile gradient and sequenced.Sequence Analysis and Amino Acid Analysis—Automated Edman

degradation was performed on an Applied Biosystems 477A pulsedliquid phase sequencer with on-line phenylthiohydantoin analysis us-ing an Applied Biosystems 120A HPLC system operated according tomanufacturer recommendations. Peptide concentrations were deter-mined using amino acid composition analysis. Peptide samples (approx-imately 500 pmol) were hydrolyzed for 24 h at 110 °C in 6 N HClcontaining 0.1% phenol (14). The tubes were evacuated and flushedwith nitrogen several times before they were sealed under vacuum. Thehydrolysates were analyzed in a Beckman 6300 amino acid analyzerwith an on-line Hewlett-Packard 3390A integrator and using sodiumcitrate buffers provided by the manufacturer.Fluorophore-assisted Carbohydrate Analysis—The monosaccharide

composition of the peptides was determined using FACE technology(Glyko, Novato, CA). Briefly, 200 pmol of each peptide (determined byamino acid analysis) were subjected to three separate hydrolysis reac-tions to determine the presence of NeuNAc (0.2 N TFA, 80 °C 3 1 h),amine sugars (8 N TFA, 100 °C 3 3 h), and neutral sugars (4 N TFA,100 °C 3 5 h). The released monosaccharides were then labeled over-night at 37 °C with a fluorescent label as described by the manufac-turer, and the labeled monosaccharides were resolved on a proprietarygel system. The gels were imaged using a fluorescence camera linked toa computer, and data were analyzed using proprietary software.Electrospray Ionization-Mass Spectrometry (ES-MS)—Measure-

ments were made on a Fisons VG Quattro-BQ triple quadrupole mass

spectrometer equipped with a pneumatically assisted electrostatic ionsource operating at atmospheric pressure and controlled using theMassLynxTM data system (Version 2.0). The glycopeptides, isolated asdescribed above, were lyophilized and resuspended in aqueous acetoni-trile (50%) containing 1% formic acid. Spectra were acquired in themulti-channel acquisition mode from mass/charge (m/z) 600-1600 witha scan time of 10 s. For some experiments, the reversed phase fractionswere resubjected to reverse phase-HPLC (Deltabond, ODS 150 3 1 mm,Keystone Scientific, Bellefonte, PA) using an Isco (Lincoln, NA) micro-bore system. The effluent was monitored at an absorbance of 216 nmand split evenly into two streams. One stream was fed directly to the ionsource of the mass spectrometer. Spectra were acquired in continuummode 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 athalf-height of 1.0 Da form/z 893. The mass spectra were transformed toa molecular mass using software supplied by the manufacturer.

RESULTS

HPLC Separation of Glycopeptides—The glycopeptides puri-fied by jacalin-agarose affinity chromatography resolved intothree 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 peptideeluting at 20% (pep1) acetonitrile is novel. Edman degradationamino acid sequence analysis of the first 20 amino acid resi-dues of peptides 2 and 3 confirmed that both of these peptideswere derived from Pg 2 and consisted of a fragment commenc-ing at Ile-329 (Table I). Confirmation of the sequence of the last18 residues was obtained by further digesting peptides 2 and 3with SV8 as described under “Experimental Procedures.” Theidentity of pep1 was obtained by amino acid sequencing of theentire tryptic peptide. The sequences (Table I) differed slightlyfrom the Pg sequence previously published. No phenylthiohy-dantoin amino acid derivative was detected in the cycle duringEdman degradation at position Thr-345 in peptides 2 and 3,consistent with a modification of the threonine by the knownO-linked carbohydrate chain present at this residue (3). Ser-338 in peptide 2 was also modified and both peptides 2 and 3

FIG. 1. Chromatograph of HPLC of jacalin-agarose affinity pu-rified tryptic peptides of Pg 2. The chromatograph shows the elutionprofile of a C18 column. Peptides were resolved using a 0–40% aceto-nitrile gradient. All buffers were 0.1% TFA. The flow rate was 0.5 mlmin21. Peptides were monitored by measuring the absorbance at awavelength of 214 nm.

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had Gln-341 instead of the expected Glu-341, as reported pre-viously (12). Peptides 2 and 3 always eluted from the C18column in equimolar concentrations as indicated by peak area(data not shown).Amino acid sequence analysis of peptide 1 (Table I) indicated

that it was a Pg 2 derived fragment, commencing at Cys-242and terminating at Lys-257. There was a blank cycle at positionSer-248 in this peptide tide, indicating a modification of theserine. The amount of peptide 1 obtained from HPLC wasconsistently 13–15% of the amounts of peptides 2 or 3, suggest-ing that not all Pg 2 molecules have this modification.Mass Spectrometry Analysis—To determine the nature of the

modification inferred by the blank cycles found during se-quence analysis, we performed ES-MS analysis. ES-MS analy-sis of peptide 1 (Table II) revealed a mass of 2449.9 Da. Theexpected mass of the unmodified carboxyamidomethylated pep-tide is 1796.0 Da. As the predicted mass of a mucin carbohy-drate trisaccharide chain attached to the side chain of serine orthreonine is 656.6 Da, this indicates that Ser-248 may possessan O-linked mucin carbohydrate chain.Similar analysis of peptide 2 revealed a mass of 4896.6 6 1

Da (Table II). The expected mass of the carboxyamidomethyl-ated peptide is 4162.5. Mass spectrometry analysis of peptide 3consistently revealed a peptide with mass value of 4816.2 6 0.6Da (Table II). Thus, peptides 1, 2, and 3 all have a greater massthan would be expected from a simple analysis of the primarysequence, indicating that all peptides isolated from jacalin-agarose are modified by the addition of at least one trisaccha-ride moiety on Ser or Thr. To confirm the composition andstructure of the carbohydrate on these glycopeptides we per-formed FACE analysis.FACE Analysis—Peptides 1, 2, and 3 were analyzed by

FACE to determine carbohydrate composition. A representa-tive analysis is shown in Fig. 2. The only monosaccharidesdetected in peptides 1, 2, and 3 were galactose, N-acetylgalac-tosamine, and NeuNAc. The ratios of N-acetylgalactosamine toprotein (Table III) are essentially equimolar for peptides 2 and3, indicating that the additional modification of Ser-338 notedin peptide 2 is unlikely to be a carbohydrate modification.Desialylation of Glycopeptides—Glycopeptides 1–3 were

treated with NANase III, a neuraminidase with specificity fora(2–3), a(2–6), and a(2–8) linkages for 16 h at 37 °C. Thepeptides were then analyzed by ES-MS. The mass differencesbefore and after NANase III treatment are consistent with theloss of the NeuNAc (Table IV), indicating that each of theglycopeptides purified only has one NeuNAc residue.

DISCUSSION

In this study, we provide evidence that Pg 2 molecules con-tain an additional O-linked carbohydrate chain and have local-ized the site of attachment of this second carbohydrate chain toSer-248. We have isolated three jacalin-reactive peptides fromPg 2, of which one (peptide 1) is a novel glycopeptide. The othertwo peptides, designated peptides 2 and 3, have been describedpreviously (12). The mass spectrometry and carbohydrate com-position analysis of jacalin-purified peptide 1 indicates that thecarbohydrate attached to Ser-248 of Pg 2 has the structure

NeuNAca2–3Galb1–3GalNAc, identical to the known structureof 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, andthat regulate the activation of Pg as well as plasmin activity.The site of attachment for the novel sialylated trisaccharidechain described here is located between kringles 2 and 3. Weand others (6, 8) have demonstrated the importance of carbo-hydrate in the regulation of Pg activation by tPA. We have alsopreviously demonstrated that a decrease in the catalytic effi-ciency of Pg activation by tPA correlates with increasing Neu-

FIG. 2. FACE gel analysis of monosaccharides present in pep-tide 1. Lane 1, 100 pmol of fluorescently labeled monosaccharides (aslabeled); lane 2, neutral monosaccharides on peptide 1; lane 3, aminemonosaccharides present on peptide 1; lane 4, 200 pmol of fluorescentlylabeled N-acetylneuraminic acid; and lane 5, NeuNAc present on pep-tide 1. In all cases, 75–200 pmol of peptide were hydrolyzed. The glucoseseen in lanes 2, 3, and 5 is a natural contaminant in this detectionsystem.

TABLE IIMass values of peptides 1, 2, and 3 derived from ES-MS analysisThe mass of the trisaccharide is predicted on the basis of the meas-

ured mass of the glycopeptide and that for the peptide predicted on thebasis of the sequence data alone. Under these constraints, the differ-ence will be the computed mass of the trisaccharide (675 Da) minus thecontribution of water (18 Da). The added average mass of NeuNAca2–3Galb1–3GalNAc trisaccharide (attached to the side chain of Ser) byelectrospray mass spectrometry analysis, therefore, is 656.6 Da. Thedifference between the mass predicted on the basis of electrospray massspectrometry of the glycopeptide reflects the experimental error asso-ciated with the technique (61 Da per 1 kDa). The mass values obtainedfor peptide 2 are 80 Da larger than the mass values obtained for peptide3, suggesting a further modification, possibly a phosphorylation onSer-338 (see text). Data derived as described under “ExperimentalProcedures.” Values are the means of at least three determinationsperformed on three separate peptide preparations.

Peptide ES-MS Expected mass Difference

Da Da Da

Peptide 1 2449.9 6 0.1 1796.01 653.9Peptide 2 4896.6 6 1.0 4162.5 734.1Peptide 3 4816.2 6 0.6 4162.5 653.7

TABLE IIIRatios of N-acetylgalactosamine to protein as determined by FACE

Glycopeptide pmol Peptidea pmol N-acetylgalactosamine

Peptide 2 100 150 6 6.2Peptide 3 100 122 6 11.0

a Determined by amino acid analysis. Values are mean 6 S.E., n 5 4.

TABLE IPrimary structure of glycopeptides pep1, pep2 and pep3

The cDNA sequence suggests that the blank cycle (X) detected following Edman degradation in peptide 1 at position 248 is a serine residue. Aserine is also present in the blank cycle at position 338 in peptide 2. The blank cycle at position 345 in peptides 2 and 3 is a threonine and is knownto possess an O-linked carbohydrate chain (3, 4).

Peptide Sequence

Peptide 1 242CTTPPPXSGPTYQCLK-257

Peptide 2 329IPSCDSSPVXTEQLAPXAPPELTPVVQDCYHGDGQSYR2366

Peptide 3 329IPSCDSSPVSTEQLAPXAPPELTPVVQDCYHGDGQSYR2366

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NAc content (6, 7). It has been suggested that activation of Pgby tPA may rely on a very precise alignment of substrate andactivator (15). Thus, the presence of more than one sialylatedtrisaccharide chain between kringle domains on Pg 2 moleculesmay disrupt important protein-protein interactions, leading tothe observed reduced activation efficiency and providing astructural basis for this functional phenomenon. Table V dis-plays a summary of data derived from the literature demon-strating the correlation between NeuNAc content and decreas-ing activation efficiency by tPA.Since there are at least six glycoforms of Pg 2 (4, 5, 6), the

presence of a second sialylated trisaccharide on Ser-248 doesnot fully account for all the known microheterogeneity of thisprotein. There are other types of O-linked saccharide chainsthat can contain NeuNAc, notably oligosaccharide chains at-tached via a fucose residue (16) that are found in a variety ofproteins involved in coagulation (17). The presence of such

carbohydrate chains on plasminogen or the possibility of poly-sialic acid (6) must also be considered. Our data also demon-strate a modification of Ser-338 of plasminogen 2. Hortin andYu (18) have presented evidence indicating that plasminogen 2is phosphorylated at Ser-338. The mass difference of 80 Da wereport here between peptides 2 and 3 provides further evidencein support of this hypothesis.In conclusion, we provide evidence for a novel O-linked car-

bohydrate chain on Pg 2. This chain, attached at Ser-248, is atrisaccharide terminated with NeuNAc. The presence of thistrisaccharide between kringles 2 and 3, coupled with thepreviously reported trisaccharide between kringles 3 and 4(3), provides a structural basis for the observed correlation ofNeuNAc content with structural and functionalmicroheterogeneity.

Acknowledgments—The authors thank Mario Gonzalez-Gronow foruseful discussion, Zuzana Valnickova for performing the Edman deg-radation amino acid sequence analysis, and Ida B. Thøgersen for per-forming the amino acid analysis. We also thank Hanne Grøn, TammyMoser, and David Morgan for critical reading of the manuscript. Specialthanks go to Sharon Stack for invaluable help with the manuscript.

REFERENCES

1. Davidson, D. J., and Castellino, F. J. (1991) Biochemistry 30, 6689–66962. Hayes, M. L., and Castellino, F. J. (1979) J. Biol Chem. 254, 8768–87713. Hayes, M. L., and Castellino, F. J. (1979) J. Biol Chem. 254, 8777–87804. Siefring, G. E., Jr., and Castellino, F. J. (1974) J. Biol. Chem. 249, 7742–77465. Gonzales-Gronow, M., Grennet, H. E., Fuller, G. M., and Pizzo, S. V. (1990)

Biochim. Biophys. Acta 1039, 269–2766. Pirie-Shepherd, S. R., Jett, E. A., Andon, N. L., and Pizzo, S. V. (1995) J. Biol.

Chem. 270, 5877–58817. Pirie-Shepherd, S. R., Serrano, R. L., Andon, N. L., Gonzalez-Gronow, M., and

Pizzo, S. V. (1996) Fibrinolysis 10, 49–538. Mori, K., Dwek, R. A., Downing, A. K., Opdenakker, G., and Rudd, P. M. (1995)

J. Biol. Chem. 270, 3261–32679. Davidson, D. J., and Castellino, F. J. (1993) J. Clin. Invest. 92, 249–25410. Edelberg, J. M., Enghild, J. J., Pizzo, S. V., and Gonzalez-Gronow, M. (1990)

J. Clin. Invest. 86, 107–11211. Gonzalez-Gronow, M., Edelberg, J. M., and Pizzo, S. V., (1989) Biochemistry

28, 2374–237712. Hortin, G. L., (1990) Anal. Biochem. 191, 262–26713. Hortin, G. L., and Trimpe, B. L. (1990) Anal. Biochem. 188, 271–27714. Meltzer, N. M., Taus, G. I., Gruber, S., and Stein, S. (1987) Anal. Biochem. 160,

356–36115. Madison, E. L., Coombs, G. S., Corey, D. R., (1995) J. Biol. Chem. 270,

7558–756216. Harris, R. J., van Halbeek, H., Glushka, J. Basa, L. J., Ling, V. T., Smith, K.

J., Spellman, M. W. (1993) Biochemistry 32, 6539–654717. Bharadwaj, D., Harris, R. J., Kisiel, W., and Smith, K. J. (1995) J. Biol. Chem.

270, 6537–654218. Hortin, G. L., and Yu, H. (1994) Blood 84, Suppl. 1, 193 (abstr.)

TABLE VActivation efficiency of isotypes of plasminogen 2 by tissue type

plasminogen activator

Isoform NeuNAc contenta Activator Catalytic efficiency of activationb

mol/mol protein M21 min21

Pg2a 1.3 6 0.317 tPA 3.84 3 106

Pg2b 2.2 6 0.201 tPA 1.78 3 106

Pg2g 2.95 6 0.035 tPA 2.72 3 106

Pg2d 5.77 6 2.50 tPA 2.85 3 106

Pg2e 5.34 6 0.68 tPA 1.71 3 106

Pg2f 13.65 6 4.07 tPA NDc

a Data are derived from Ref. 6.b Data are derived from Ref. 7.c ND, not detectable.

TABLE IVDesialylation of glycopeptides from Pg 2 by NANase III

Data are derived from ES-MS analysis. Desialylation was performedas described under “Experimental Procedures.” The predicted loss ofmass due to desialylation is 291.3 Da. The technique has an expectederror of 6 1 Da per 1 kDa.

Glycopeptide Peptide 1 Peptide 2 Peptide 3

Mass before desialylation (Da) 2449.9 4896.6 4816.2Mass after desialylation (Da) 2141.3 4604.1 4526.0Difference (Da) 308.6 292.5 290.2

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Salvatore V. PizzoSteven R. Pirie-Shepherd, Robert D. Stevens, Nancy L. Andon, Jan J. Enghild and

Plasminogen 2-Linked Sialylated Trisaccharide on Ser-248 of HumanOEvidence for a Novel

doi: 10.1074/jbc.272.11.74081997, 272:7408-7411.J. Biol. Chem. 

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