NovelCarbohydrate-bindingActivityofPancreaticTrypsins … ·...

Novel Carbohydrate-binding Activity of Pancreatic Trypsinsto N-Linked Glycans of Glycoproteins*

Received for publication, December 27, 2005 Published, JBC Papers in Press, January 17, 2006, DOI 10.1074/jbc.M513773200

Hiroko Takekawa‡, Chieko Ina‡, Reiko Sato§, Kazunori Toma§, and Haruko Ogawa‡¶1

From the ‡Graduate School of Humanities and Sciences and ¶The Glycoscience Institute, Ochanomizu University, Bunkyo-ku,Tokyo 112-8610 and §The Noguchi Institute, Itabashi-ku, Tokyo 173-0003, Japan

How glycosylation affects the reactivity of proteins to trypsin isnot well understood. Bovine and porcine pancreatic trypsins werediscovered to bind to�-Man,Neu5Ac�2,6Gal�1,4Glc, and�-galac-tose sequences by binding studies with biotinylated sugar-poly-mers. Quantitative kinetic studies supported that phenylmethylsul-fonyl fluoride (PMSF)-treated trypsin binds to glycolipid analoguespossessing �-Man or �-NeuAc but not to those possessing �-galac-tose or �-GlcNAc residue. Enzyme-linked immunosorbent assay(ELISA) showed that trypsin binds to six kinds of biotinylated gly-coproteins possessing highmannose-type and complex-typeN-gly-cans but not to bovine submaxillary mucin, which possesses onlyO-glycans. Further, the binding of trypsin to glycoproteins was dif-ferentially changed by treatments with sequential exoglycosidases,endoglycosidase H, orN-glycosidase F. Quantitative kinetic studiesindicated that PMSF-treated trypsin binds with bovine thyroglobu-lin with the affinity constant of 1010 M�1, which was the highestamong the glycoproteins examined, and that �-galactosidase treat-ment decreased it to 105 M�1. PMSF-treated trypsin bound to otherglycoproteins, including ovomucoid, a trypsin inhibitor, with theaffinity constants of 108-105 mol�1 and were markedly changed byglycosidase treatments in manners consistent with the sugar-bind-ing specificities suggested by ELISA. Thus, the binding site for gly-cans was shown to be distinct from the catalytic site, allowing tryp-sin to function as an uncompetitive activator in the hydrolysis of asynthetic peptide substrate. Correspondingly the carbohydrate-binding activities of trypsin were unaffected by treatment withPMSForsoybeantrypsin inhibitor.Theresults indicate thepresenceofan allosteric regulatory site on trypsin that sugar-specifically interactswith glycoproteins in addition to the proteolytic catalytic site.

Numerous biological phenomena aremediated by recognition of spe-cific oligosaccharide signals. This recognition implies quality control inpolypeptide folding, cellular interactions, and protein targeting (1–3). Incontrast, some functions of protein glycosylation seem to be widelyapplicable to various types of glycosylation, for example, protectingagainst proteolysis, stabilizing active conformations, and affording sol-ubility to proteins (3). These functions have been attributed to ambig-uous steric effects of glycosylation in the absence of clear structuralspecificity, but the involvement of glycan recognition in achieving thesefunctions has not yet been elucidated. Clarification of the molecular

mechanism by which glycosylation plays a role in protecting or stabiliz-ing the active conformation of proteins would enable the use of glyco-sylation in molecular engineering of recombinant products for thera-peutic purposes.Trypsin is a principal pancreatic serine protease that plays a key role

in digestion in the duodenum by activating zymogens and degradingdietary proteins. Trypsin acts specifically on peptide bonds of the car-boxyl side of positively charged lysine and arginine and catalyzes theactivation of many pancreatic proenzymes, such as trypsinogen, chy-motrypsinogen, proelastase, and carboxypeptidase, and protease-acti-vated receptors to control digestive efficiency in the intestines (4, 5).When, however, trypsin is activated in the pancreas, the activated pro-teinases induce the destruction of pancreatic cells. The modulation oftrypsin activity is therefore important for controlling digestive effi-ciency and preventing pancreatitis.Porcine pancreatic �-amylase (PPA)2 is activated by interaction with

glycoproteins. Previously we reported that PPA exhibits carbohydrate-binding activity toward N-glycans of glycoproteins (6). To further elu-cidate the biological functions of the carbohydrate-binding activityfound in PPA, we investigated whether other pancreatic digestiveenzymes possess similar carbohydrate-binding activity. In this study, wefound that trypsin exhibits remarkable carbohydrate-binding activitiesto the sequences present in the N-glycans of glycoproteins with a spec-ificity distinct from PPA. This finding provides new insights into theinteraction between the proteases and glycoproteins related to proteaseresistance and the biological functions of carbohydrate-specific interac-tions in the digestive organs.

EXPERIMENTAL PROCEDURES

Materials—Porcine pancreatic trypsin (PPT), N-�-benzoyl-L-argi-nine ethyl ester (BAEE), N-�-benzoyl-DL-arginine-p-nitroanilidehydrochloride (BAPA), soybean trypsin inhibitor, bovine serum albu-min (BSA), 3,3�-diaminobenzidine tetrahydrochloride, methyl-�-D-mannoside, and mannitol were purchased from Wako Pure ChemicalIndustries, Ltd., Osaka, Japan. Bovine pancreatic trypsin (BPT), bovinesubmaxillary gland mucin (BSM), human holotransferrin, fetuin fromfetal calf serum, hen ovomucoid, human orosomucoid, bovine thyro-globulin, streptavidin-biotinylated horseradish peroxidase complex(ABC complex), and 4-nitrophenyl phosphate magnesium salt werepurchased from Sigma. Sugar-biotinylated polyacrylamide probes (sug-ar-BP probes) were purchased from Lectinity Holdings, Inc. Moscow,

* This work was supported in part by Grants-in-aid for Scientific Research (C) 14580622and 17570109 (HO) from the Japan Society for the Promotion of Science and Grants-in-aid for Scientific Research on Priority Areas 15040209 and 17046004 (HO) from theMinistry of Education, Culture, Sports, Science, and Technology. The costs of publica-tion of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

1 To whom correspondence should be addressed. Tel.: 81-3-5978-5343; Fax: 81-3-5978-5343; E-mail: [email protected].

2 The abbreviations used are: PPA, porcine pancreatic �-amylase; BPT, bovine pancreatictrypsin; PPT, porcine pancreatic trypsin; BAEE, N-�-benzoyl-L-arginine ethyl ester;BAPA, N-�-benzoyl-DL-arginine-p-nitroanilide hydrochloride; BSA, bovine serumalbumin; BSM, bovine submaxillary mucin; ABC complex; streptavidin-biotinylatedhorseradish peroxidase complex; sugar-BP probe, sugar-biotinylated polyacrylamideprobe; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris-buffered saline; KA, affinity con-stant; ka, association rate constant; kd, dissociation rate constant; ELISA, enzyme-linked immunosorbent assay; SPR, surface plasmon resonance; RU, resonance units;Me, methyl.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 13, pp. 8528 –8538, March 31, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Russia, except �-D-galactose-3-sulfate, which was purchased fromSeikagaku Corp., Tokyo, Japan. Porcine thyroglobulin and Galanthusnivalis lectin were purchased from Cosmo Bio Co., Ltd, Tokyo, Japan.Neuraminidase fromVibrio cholerae,N-glycosidase F from Flavobacte-riummeningosepticum, andO-glycosidase were purchased from RocheDiagnostics Corp., Inc. (Indianapolis, IN). Psathyrella velutina lectinwas prepared in our laboratory (7).�-Galactosidase and�-N-acetylhex-osaminidase from jack bean and �-galactosidase fromMortierella vina-cea were purchased from Seikagaku Corp. EZ-Link sulfo-N-hydroxy-succinimide-biotin and Sambucus nigra bark lectin were purchasedfrom Funakoshi Co. Ltd., Tokyo, Japan. Phenylmethylsulfonyl fluoride(PMSF) and methyl-�-D-galactoside were purchased from NacalaiTesque, Inc., Kyoto, Japan.N-Heptyl-�-D-thioglucoside was purchasedfrom Dojindo Laboratories, Kumamoto, Japan. Peanut lectin and Rici-nus communis agglutinin I were purchased from Seikagaku Corp. Lac-tose was purchased from Kanto Kagaku, Tokyo, Japan. SDS-PAGEmolecular weight standards were purchased from Bio-Rad.

Preparation of Glycoprotein Probes—All biotinylated glycoproteinprobes, their deglycosylated derivatives, and biotinyl lectins were pre-pared in our laboratory. Biotinylation was performed using EZ-linkTM

sulfo-N-hydroxysuccinimide-biotin according to the instruction man-ual. Briefly, 2 mg of each glycoprotein was dissolved in 1 ml of 50 mM

sodium bicarbonate buffer (pH 8.5), and 74 �l of sulfo-NHS-biotin (1mg/ml) was added. After incubation for 30 min at room temperature,the reactant was dialyzed against water to remove excess biotin. Asialo-glycoproteins, asialoagalactoglycoproteins, and asialoagalactoahex-osaminoglycoproteins were prepared from biotinylated glycoproteinsby sequential glycosidase treatments with neuraminidase (0.1 units/mgglycoprotein) in 20mMacetate-buffered saline (pH 5.5),�-galactosidase(0.14 units/mg glycoprotein) in 50 mM sodium-citrate buffer (pH 3.5)overnight, and then�-N-acetylhexosaminidase (1.43 units/mg of glyco-protein) in 50mM sodium citrate buffer (pH 5.0) at 37 °C overnight. Theglycan structures and carbohydrate concentrations of the glycoproteinsused in this study are summarized in Scheme 1. Besides the sequentialtreatments described above, biotinylated bovine thyroglobulin wastreated with �-galactosidase (0.14 unit/mg of glycoprotein) in 20 mM

acetate-buffered saline (pH 5.5) for agalactosylation of the major oligo-saccharide, Gal�1–3Gal�1–4GlcNAc. Fetuin was treated with N-gly-cosidase F (600 units/mg of glycoprotein) in 10 mMTris-buffered saline(TBS) at pH 7, or de-O-glycosylated with a mixture of neuraminidase

SCHEME 1. Major oligosaccharide structures ofglycoproteins used in this study.

Carbohydrate Binding Activity of Pancreatic Trypsins

MARCH 31, 2006 • VOLUME 281 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8529


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(0.1 unit/mg of glycoprotein) andO-glycosidase (2 milliunits/mg of gly-coprotein) in 10 mM acetate buffer of pH 6 at 37 °C overnight.Biotinylated porcine thyroglobulin (20 �g) was denatured in glycopro-

tein denaturing buffer (5% SDS, 10% �-mercaptoethanol) at 100 °C for 10min, a 10% volume of 0.5 M sodium citrate buffer (pH 5.5) was added, andthen it was incubated with 1500 units of endoglycosidase H at 37 °C over-night. Deglycosylation of all biotinylated glycoprotein probes was checkedby ELISA for a change in reactivity with Ricinus communis agglutinin I foragalactosylation, Psathyrella velutina lectin for desialylation and ahex-osaminylation, Galanthus nivalis lectin for endoglycosidase H treatment,and peanut lectin for de-O-glycosylation to recognize each carbohydratestructure, and by mobility on SDS-PAGE to demonstrate the decrease inmolecular weight (data not shown).

SDS-PAGE—To check the purity of trypsin, SDS-PAGE was per-formed according to the method of Laemmli (8) using a 14% gel in thepresence of 2-mercaptoethanol. The bovine and porcine trypsins (10,20, or 40 �g of protein per lane) were loaded onto the gel together witha set of markers and run at 20 mA for 1.5 h. After electrophoresis,protein bands were visualized by Coomassie Brilliant Blue R-250.

Binding Studies with Sugar-BP Probes or Biotinylated GlycoproteinProbes—PPTandBPTwere preincubated in the presence or absence of 0.5mMPMSF, 0.5mMsoybean trypsin inhibitor, or 5mMEDTAin10mMTBS(pH7.5) for1hand then immobilizedat concentrationsof0.01–0.5�g/100�l inwells of amicrotiter plate (Immulon 1,Dynatech Laboratories) at 4 °Covernight. All other procedures were performed at room temperatureusing 10mMTBS (pH 7.5) as the dilution buffer. After immobilization, thewells were blocked with 3% BSA for 2 h. Aliquots (100 �l) of various sug-ar-BP probes (shown in Scheme 1A) or biotinylated glycoprotein probes atconcentrationsof10�g/mlwereadded toeachwell, followedby incubationfor 1 h. After incubation, the wells were washed three times, and 100 �l ofABC complex (1�g/ml) was added, and themixturewas incubated for 1 h.After washing three times, color was developed by adding 200�l of o-phe-nylenediamine/H2O2, and then 50�l of 2.5MH2SO4was added to stop thereaction. Absorbance was measured with a microplate reader (Bio-RadMPR-80) at 490 nm.

Quantification of Interactions between Trypsins and Glycolipid Ana-logues by Surface Plasmon Resonance—For binding studies betweentrypsins and glycolipid analogues or various glycoproteins, a BIAcore2000 SPR apparatus (BIAcore AB, Uppsala, Sweden) was used. Struc-ture of glycolipid analogues used in this study was illustrated in Scheme2B, and their synthesiswill be described elsewhere.Glycolipid analogueswere immobilized on a HPA sensor chip (BIAcore AB) by preparingliposomes containing each glycolipid analogue/phosphatidylcholine atamolar ratio of 40/60 as described previously (9, 10). PMSF-treated PPT

was injected onto the sensor chip at various concentrations in 10 mM

TBS buffer (pH 7.5) at a flow rate of 20 �l/min at 25 °C using a BIAcorebiosensor. The reference cell was prepared by immobilizing phosphati-dylcholine and used to correct for bulk effect. The chip was regeneratedeach time by injection of 20 �l of 0.1 M phosphoric acid.

Quantification of Interactions between Trypsins and Various Glyco-proteins by SPR—After equilibration of a CM5 sensor chip (BIAcore AB)with HEPES-buffered saline, the surface of the sensor chip was activatedwith an amine coupling kit. BPT or PPT (each 1.8 �M) in 10 mM sodiumacetate buffer (pH 6) containing 0.1 mM PMSF and 0.2 M methyl �-D-mannosidewas injected onto the activated surface, and then the remainingN-hydroxysuccinimide esters were blocked with 1.0 M ethanolaminehydrochloride (pH 8). Each step was performed for 14 min at a constantflow rate of 10 �l/min at 25 °C. The reference flow cell was prepared withBSA as a ligand.To determine the pH dependence of the binding, fetuin, or porcine thy-

roglobulin were dissolved at 30 �g/ml in buffers of various pH, 10 mM

acetate (pH 4.5, 5.5, and 6.5), 10 mM TBS (pH 7.0, 7.5, and 8.0), or 10 mM

bicarbonate buffer (pH 9 and 10), and injected onto the trypsin-immobi-lized sensor chip. To measure binding curves, various glycoproteins in 10

FIGURE 1. Reactivities of PPT (A) and BPT (B) toward sugar-BP probes by ELISA. PPTand BPT (100 �l) were coated onto the wells of a microtiter plate and reacted with varioussugar-BP probes as described in the text. The bound sugar-BP probes were detected withABC complex and o-phenylenediamine/H2O2 by ELISA. Symbols used are: �, �-Man-BP;�, �-Man-6-phosphate-BP and Neu5Ac �2– 6Gal�1– 4Glc-BP; �, �-Gal-BP; �, �-Glc-BP;‚, �-GalNAc-BP; Œ, Neu5Ac �2–3Gal�1– 4Glc-BP; ‰, �-Neu5Ac-BP; , �-Glc-BP; f, Glc-NAc�1– 4GlcNAc-BP;

�� , GlcNAc�1– 6(Gal�1–3)GalNAc (core2)-BP, �-GalNAc-, �-Gal-,

LacNAc-, Lac-, �-GlcNAc-, and �-Gal-3-sulfate-BP. The label “core 2*” stands for the sevensugar-BP probes that bound very little with trypsin.

SCHEME 2. Structures of sugar-BP probes and glycolipid analogues used in this study.




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mM TBS (pH 7.5) were separately injected onto the trypsin-immobilizedsensor chip at concentrations of 1, 0.5, 0.25, 0.125, and 0.0625�M for 150 sat a flow rate of 20�l/min at 25 °C. The chip was regenerated each time byinjection of 20�l of 10mMHCl. To assay inhibition, a trypsin-immobilizedsensor chip was equilibrated with TBS containing 50 mM or 0.2 Mmethyl-�-mannoside, methyl-�-galactoside, or lactose for 15 min, and then theglycoprotein dissolved in the same buffer was injected. Kinetic parameterswere calculated mainly by global analysis, or affinity analysis when neces-sary, using the BIAevaluation software version 3.1.

Measurement of Enzyme Activity of Trypsin—Enzyme activity wasmeasured in a test tube according to themethodpreviously describedusingBAEE (11) or BAPA (12) as the substrate. To estimate the effect of sugar ontheBAEE-hydrolytic activity, itwasmeasuredafterpreincubationofPPT in

100�l of 1mMHCl (0.02mg/ml) with 100�l of 0.2 Mmethyl-�-D-manno-side, 0.2 Mmethyl-�-D-galactoside, or 0.2 M lactose in 20mMTBS (pH 7.6)for 10 min at 25 °C. A control experiment was done by preincubating PPTwithout sugar.Afterpreincubation, thePPTsolutionwasadded to500�l of0.025 mM BAEE in 10 mM TBS (pH 7.6) and incubated at 25 °C for 1 min.Absorbancewas immediatelymeasured at 253nm, and trypsin activity thatincreasedabsorbanceby0.003at25 °C for1minwas regardedas1USPunitof trypsin.To analyze the effect of sugar by a double reciprocal Lineweaver-Burk

plot, the initial rates of the enzyme-catalyzed reaction were measuredusing BAPA as the substrate. The substrate stock solution was preparedby dissolving 0.0217 g of BAPA in 1.5 ml of Me2SO and then diluted tofinal concentrations of 0.1–0.5 mM with 100 mM TBS (pH 7.5) in thepresence or absence of 0.2 M methyl-�-mannoside, methyl-�-galacto-

FIGURE 2. Quantification of interaction betweenPPT and glycolipid analogues by SPR. Glycolipidanalogues were immobilized on an HPA sensor chipas described in the text. PPT was pretreated with 0.1mM PMSF and injected onto the sensor chip at vari-ous concentrations in 10 mM TBS buffer (pH 7.5) at aflow rate of 20 �l/min at 25 °C using BIAcore. Bindingcurves of PPT on the sensor chip immobilized withglycolipid analogues containing �-Man (A), �-NeuAc(B), and �-Lac (C) are shown. The response isexpressed as the change in the number of resonanceunits induced by the binding of PPT to the glycolipidanalogue-immobilized flow cell, which was cor-rected for bulk effect by subtracting the change onthe phosphatidylcholine-immobilized reference cell.D, kinetic parameters for the interaction betweenPPT and glycolipid analogues. Kinetic parameterswere calculated by global analysis for �-Man andaffinity analysis for �-NeuAc and �-Lac. ka, associa-tion rate constant; kd, dissociation rate constant; KA,association constant.

FIGURE 3. SDS-PAGE of BPT and PPT. Bovine and porcine trypsin (10, 20, or 40 �g ofprotein per lane) were loaded under reduced condition onto a 14% polyacrylamide gel.SDS-PAGE was performed as described in the text, and protein bands were visualized byCoomassie Brilliant Blue R-250. The migration positions of molecular weight markers areshown on the left side of the gel.

FIGURE 4. Reactivities of PPT (A) and BPT (B) to biotinylated glycoproteins by ELISA.PPT or BPT (each 100 �l) was coated onto the wells of a microtiter plate and reacted withvarious glycoprotein probes as described in the text. The bound glycoprotein probeswere detected with ABC complex and o-phenylenediamine/H2O2 by ELISA. Symbolsused are: �, bovine thyroglobulin; �, porcine thyroglobulin; Œ, fetuin; f, ovomucoid;�, orosomucoid; �, transferrin; and

�� , BSM.




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side, or lactose. The substrate solutions were heated for 5 min at 37 °C.The enzyme stock solution was prepared by dissolving 2.5 mg of PPT in3 ml of 1 mM HCl containing 13 mg of CaCl2-2H2O. A 10-�l aliquot ofthe enzyme solution was added to 3 ml of the substrate solution, andabsorbance was measured at 410 nm every 30 s.

RESULTS

Interaction between Trypsins and Sugar-BP Probes

The carbohydrate-binding activities of BPT and PPT were analyzedusing synthetic sugar-BP probes (Scheme 2A), and specificities towardsugar residues and oligosaccharides were determined. As shown in Fig. 1,bothBPTandPPTexhibitedhighbinding activity toward�-Man-,�-Man-6-phosphate-, NeuAc�2,6Gal�1,4Glc-, �-Gal-, and �-Glc-BP probesamong the 17 kinds of sugar-BP probes tested. Both trypsins bound toNeuAc�2,3Gal�1,4Glc- and NeuAc-BP to a lesser extent than toNeuAc�2,6Gal�1,4Glc-BP, indicating a preference for sialyl linkages. Onthe contrary, trypsindidnot bindwith�-GalNAc-ormucin core 2-typeBPprobes or with �-Gal, LacNAc, Lac, �-GlcNAc, or �-Gal3-sulfate-BP

probes. None of the carbohydrate-binding activities of the trypsins wasaffected by preincubationwith PMSF and EDTAor soybean trypsin inhib-itor, suggesting that binding is independent of the catalytic site. The boundsugar residues other than �-D-Glc are components of N-glycans, demon-strating basic specificities toward monosaccharides or short sequencesthat include linkages for trypsin binding.

Interaction between Trypsin and Glycolipid Analogues Analyzed by SPR

To verify the carbohydrate-binding specificity of trypsin by quantita-tive measurement, interaction analyses were performed by SPR usingfive kinds of synthetic high sensitivity glycolipid analogues (Scheme 2B).The total amounts of immobilized glycolipid analogues containing�-Man, �-NeuAc, and �-Lac were 1593, 1560, and 1680 BIAcore reso-nance units (RU, 1000 RU � 1 ng/mm2), respectively. As shown in Fig.2 (A–C), PPT concentration-dependently bound to immobilized glyco-lipid analogues. The differential binding of PPT to the analogues clearlyindicates its relative binding affinity toward sugar residues: PPT bindsbest with the analogues containing �-Man, then �-NeuAc, and to alesser extent �-Lac. The binding and dissociation occurred rapidly atthe start and end of injection of the glycolipid analogues except the�-Man derivative, demonstrating the specific binding of PPT to thoseglycolipid analogues with quick association and dissociation rates. PPTdid not bind to other analogues containing �-galactose or �-GlcNAceven at 2 �M. The association constants (KA) were calculated to be106-105 (M�1) for glycolipid analogues containing �-Man and �-NeuAc(Fig. 2D), which are comparable to the KA obtained for the interactionbetween ricin and the glycolipid analogues containing �-galactose3 andhigher than that obtained between concanavalin A and �-Man-derivat-ized glycolipid containing phosphatidylethanolamine aglycon (13). Thecarbohydrate-binding specificity indicated by SPR corresponded withthat obtained by using sugar-BP probes (Fig. 1) demonstrating that theaffinity of trypsin for specific carbohydrates is comparable to those ofknown plant lectins. Therefore, the binding activity of trypsin for glyco-proteins was examined.

Purity of BPT and PPT on SDS-PAGE

The carbohydrate-binding activities shown in Figs. 1 and 2 have neverbeen reported for trypsin. To eliminate the suspicion that some contami-nant in the trypsin preparationmight exhibit such an activity, we analyzedthe purity of the trypsin preparations used in this study by SDS-PAGE. Asshown in Fig. 3, both BPT and PPT showed only a single band without anydetectable contamination even if 40 �g of trypsin preparation was appliedper lane to the polyacrylamide gel. Therefore, the carbohydrate-bindingactivities observed in this study were attributed to the trypsins.

3 H. Takekawa, C. Ina, R. Sato, K. Toma, and H. Ogawa, unpublished results.

FIGURE 5. Reactivities of BPT to biotinylated glycoprotein probes before and afterglycosidase treatment by ELISA. Biotinylated glycoprotein probes were pretreatedwith various exoglycosidases as described in the text. BPT (100 �l) was coated onto thewells of a microtiter plate and reacted with biotinylated glycoprotein probes: bovinethyroglobulin (A), porcine thyroglobulin (B), fetuin (C), ovomucoid (D), orosomucoid (E),and transferrin (F). The bound glycoprotein probes were detected by ELISA as describedin the text. Symbols used are: f, intact; E, asialo-treated; ‚, asialoagalacto-treated; �,asialoagalactoahexosamino-treated; �, �-galactosidase-treated; letter “x” with a verticalline, endoglycosidase H-treated; and �, de-N-glycosylated glycoproteins.

FIGURE 6. Binding of trypsin to fetuin and por-cine thyroglobulin at various pH by SPR. PPTand BPT were immobilized on a CM5 sensor chip,and each glycoprotein was injected onto the sen-sor chip at various pH, as described in the text.Fetuin (f) or porcine thyroglobulin (E) was dis-solved at concentrations of 1 or 0.5 �M, respec-tively, in 10 mM acetate buffer (pH 4.5– 6.5), 10 mM

TBS (pH 7– 8), or 10 mM bicarbonate buffer (pH9 –10) and injected onto the sensor chip. Thebound amounts of glycoprotein are expressed asrelative response (%) by taking the response at pH7.5 as 100%. A, relative response on immobilizedPPT; B, relative response on immobilized BPT.




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Interaction between Trypsins and Biotinylated Glycoprotein Probes

Interactions between the glycoprotein probes and PPT or BPT werestudiedbyELISAatpH7.5,which is thephysiological pH in theduodenum.Because the amount of biotin incorporated into each glycoprotein probewas almost equal, as judged by the color intensity of each probe developedwith the ABC complex being within a 10% error, the value of A490 corre-sponds to the amount of probe bound. As shown in Fig. 4(A and B), BPTand PPT were found to bind to various glycoproteins with very similarbinding patterns. The trypsins bound best to bovine thyroglobulin and to alesser extent to fetuin, porcine thyroglobulin, ovomucoid, orosomucoid,and transferrin, in that order, but not to BSM. All the bound glycoproteinscontain 5–30% (w/w) N-linked oligosaccharides, whereas BSM possessesup to 60% (w/w) O-linked glycans, which are mainly sialyl-Tn and core3-type (Scheme 1). Combined with the finding that trypsins did not bindwith �-GalNAc- and �-GlcNAc (Fig. 1), this indicates that trypsins do notinteract withO-linked glycans.The involvement of the N-glycan structure in the binding with PPT

and BPT was shown by deglycosylating the glycoprotein probes with

endo-type glycosidases. As shown in Fig. 5C, de-N-glycosylation offetuin by N-glycosidase F treatment markedly decreased the reactivitytoward both trypsins (data not shown for PPT), showing that their bind-ing was mostly due to the affinity for the sialylated complex-typeN-gly-cans of fetuin. The reactivity of trypsins for porcine thyroglobulin (Fig.5B), which contains almost equal amounts of high Man-type and com-plex-type glycans (Scheme 1), was decreased to about half that of intactporcine thyroglobulin by endoglycosidase H treatment as well as asia-loagalactoahexosaminylation, indicating that trypsins bind with highMan-type N-glycans as well as the sialylated complex-type.

As shown in Fig. 5, the effects of exo-type glycosidase treatments of theglycoprotein probes illustrate the contribution of each sugar residue to theinteraction with BPT. Remarkably, binding of BPTwith bovine thyroglob-ulin was found to be diminished by �-galactosidase treatment (Fig. 5A),clearly indicating that the�-galactosyl residue at thenonreducing terminal,which is unique to theN-glycan of bovine thyroglobulin (Scheme 1), is anepitope for BPTbinding.Other exoglycosidase treatments of bovine thyro-globulin did not affect the binding.

FIGURE 7. Effect of sugars on interactionbetween trypsins and glycoproteins. Trypsinswere immobilized on the sensor chip and preincu-bated with 50 mM or 0.2 M methyl-�-D-mannoside,lactose, or methyl-�-D-galactoside, and then theglycoprotein solution in 10 mM TBS (pH 7.5) or TBScontaining each sugar at a concentration of 50 mM

or 0.2 M was injected onto the immobilizedtrypsins. A, the binding curves of glycoproteins toPPT, and B, the binding curves of glycoproteins toBPT. Solid line, control without sugar; light dashedline, 50 mM methyl-�-D-mannoside; heavy dashedline, 0.2 M methyl-�-D-mannoside; light dotted line,50 mM methyl-�-D-galactoside; heavy dotted line,0.2 M methyl-�-D-galactoside; and dashed and dot-ted line, 50 mM Lac.




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On the other hand, neuraminidase treatment of other glycoproteinsthat possess sialylated complex-type N-glycans, such as fetuin, ovomu-coid, orosomucoid, and porcine thyroglobulin, considerably decreasedthe binding, as shown in Fig. 5 (B–E). For these glycoprotein probes�-galactosidase treatment subsequent to desialylation did not signifi-cantly change the binding, but exposure of �-mannosyl residues of thetrimannosyl core of N-glycans by �-hexosaminidase treatment subse-quent to degalactosylation restored the binding to trypsin. These results

strongly indicate that �-NeuAc and �-mannosyl residues of complex-type multiantennary N-glycans contribute to the binding of trypsins,but �-galactose and �-GlcNAc residues of the lactosamine sequence donot. The biantennary complex type of transferrin did not show signifi-cant affinity for trypsins (Fig. 5F). As a whole, the sugar-binding speci-ficities of BPT and PPT indicated in Fig. 1 coincide with and account forthe binding specificities toward the glycoprotein probes.Preincubation of PPT and BPT with PMSF and EDTA or soybean

FIGURE 8. Quantification of interaction betweentrypsins and glycoproteins by SPR. PPT and BPTwere immobilized on a CM5 sensor chip as describedin the text. Glycoproteins were injected onto a tryp-sin-immobilized sensor chip in 10 mM TBS (pH 7.5) for150 s at a flow rate of 20 ml/min at 25 °C. Theresponse was expressed as the change of resonanceunits induced by the binding of fetuin to the trypsin-immobilized flow cell, which was corrected for bulkeffect by subtracting the change on the BSA-immo-bilized reference cell. Binding curves of glycopro-teins on the sensor chip were immobilized with PPT(A) and BPT (B).




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trypsin inhibitor before the binding studies did not affect the bindingactivities toward glycoproteins (data not shown).Moreover, the bindingof trypsin to ovomucoid, a natural inhibitor that blocks the catalytic siteof trypsin, was found to be significantly affected by glycan trimming(Fig. 5D). The observations indicate that the trypsin binding to glyco-proteins is independent of their catalytic activity.

Interaction between Trypsins and Various Glycoproteins Analyzed byBIAcore

The total amounts of immobilized PPT, BPT, and BSA were 4,012,4,028, and 4,031 RU, respectively. The responses are expressed as thechange of resonance units induced by the binding of analytes to each

FIGURE 8—continued




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flow cell, which was corrected for bulk effect by subtracting the changeon the BSA-immobilized reference cell.

pH Dependence of Interaction between Trypsin and Glycoproteins—Fetuin or porcine thyroglobulin was injected onto a trypsin-immobi-lized chip at pH 4.5–10. As shown in Fig. 6, the amounts of the glyco-proteins bound to immobilized PPT andBPT changedwithin 30% in thepH range examined, showing a maximum at around pH 7.5–8.0. Theweakly alkaline pH coincides with the enteric pH indicating that thecarbohydrate-binding activity of trypsin is optimal in the milieu of theintestine. Based on this observation, 10mMTBS at pH7.5was thereafterused for the binding studies.

Effect of Sugars on Binding of Trypsin to Glycoproteins—Fig. 7 showsthe effect of monosaccharide trypsin binding to glycoproteins. Me-�-Man and Me-�-galactoside, which showed the highest binding to tryp-sin (Fig. 1) were used as inhibitors in comparison with Lac, whichshowed relatively lower binding in the binding study with BP-sugars.The binding of both trypsins to fetuin and porcine thyroglobulins wasdecreased by 30–44% in 50mMMe-�-Man and 56–64% in 0.2MMe-�-Man, and binding to bovine thyroglobulin was decreased by 44–50%and 66–70% in 50mM and 0.2 MMe-�-Gal, respectively. The binding oftrypsins was decreased by only 10–18% even at 0.2 M Lac, indicating theweak inhibitory activity of Lac compared with those of Me-�-Man andMe-�-galactoside coinciding with the relative affinity of sugars shownby BP-sugar binding studies. The results strongly support the hypothe-sis that trypsins bind glycoproteins by sugar-specific interaction.

Kinetic Parameters for Binding between Trypsins and Glycoproteins—The sensorgrams are shown in Fig. 8 (A and B), and the binding param-eters were calculated for each glycoprotein. The binding of all glycopro-teins except BSM fit best a 1:1 binding model among the fitting modelsin global analysis. The interaction between bovine thyroglobulin andtrypsins was analyzed at concentrations lower than 0.25 �M (Fig. 8, A(panel e) and B (panel o)), because bovine thyroglobulin gave fluctuat-ing, irregular-shaped binding curves at concentrations higher than 0.5�M. The interaction between trypsins and BSM showed box-shapedbinding curves suggesting high association and dissociation rates (Fig. 8,A (panel j) and B (panel t)) and was analyzed using affinity analysis. Thekinetic data of the binding are summarized in Table 1.

Trypsin bound to the glycoproteins possessing N-glycans with signifi-cantly high affinity, KA ranging from 1010-106 M�1. In contrast, the KA forBSM was as low as 1–8 � 104 M�1, indicating very weak interaction withtrypsins. The KA for bovine thyroglobulin binding to both PPT and BPTwas 1010M�1, which is the strongest among glycoproteins and equal to thatof high affinity antibodies, followed by the KA of porcine thyroglobulin,4–5 � 107 M�1. The high KA of trypsin for bovine thyroglobulin is attrib-utable to the extremely lowdissociation rate constants (kd) (10�7-10�6 s�1)compared with those of other glycoproteins (10�4-10�3 s�1), suggestingthat thyroglobulins hardly dissociate from trypsin. The KA of trypsin forbovine thyroglobulin was markedly decreased from 1010 to 105 (M�1) by�-galactosidase treatment, indicating that�-galactose residuesareessentialfor high affinity binding to trypsin. On the other hand, theKA of trypsin forporcine thyroglobulins (4–5�107M�1)wasdecreased to15–20%by treat-ment with endoglycosidase H or neuraminidase, showing that high-Mantypes as well as sialyl residues of complex types contribute to the binding.The KA for fetuin was decreased byN-glycosidase F treatment by �10�3-fold but not byO-glycosidase treatment, indicating that the sialylated com-plex-type N-glycans of fetuin contributed absolutely to the binding, butO-glycans did not, even if they are sialylated. The results correlated wellwith the reactivities of trypsin toward intact and glycosidase-modified gly-coprotein probes by ELISA.

Effect of Sugars on Enzyme Activity of PPT

As shown in Fig. 9A, the sugars that bound to trypsins enhanced theenzyme activity to various degrees, as detected using BAEE and BAPAas substrates. The hydrolytic activity of PPT for BAEE was enhanced by1.4-fold in 0.2 M methyl-�-D-mannoside and 1.2-fold in methyl-�-D-galactoside but not enhanced in 0.2 M lactose. When we used a slowlyhydrolyzable BAPAas the substrate, PPTwas activatedwith 0.2Mmeth-yl-�-D-mannoside and methyl-�-D-galactoside to �1.2- to 1.4-fold at300–600 s (Fig. 9B). As shown in Fig. 9 (C–E) and Table 1, the Lin-eweaver-Burk plots indicate that the binding of Me-�-Man, andMe-�-galactoside uncompetitively activates PPT with increasing Vmax by 2.5-fold and Km by 2- to 2.5-fold, while binding of lactose slightly inhibitedPPT noncompetitively and uncompetitively, indicating that the bindingof carbohydrates activates the hydrolytic activity to various degrees.

TABLE 1Binding parameters for interaction between trypsins and glycoproteinsInteractions between trypsin and glycoproteins were measured in 10 mM TBS (pH 7.5) using BIAcore. Kinetics parameters were calculated by global analysis for mostglycoproteins and affinity analysis for BSM. ka, association rate constant; kd, dissociation rate constant; KA, association constant (KA � ka/ka).

ka kd KA

M�1 s�1 s�1 M�1

(A) PPTBovine thyroglobulin 3.97 � 103 2.47 � 10�7 1.61 � 1010�-Galactosidase-treated bovine thyroglobulin 7.23 � 10 5.67 � 10�5 1.28 � 105Porcine thyroglobulin 3.05 � 104 7.27 � 10�4 4.19 � 107Asialo-porcine thyroglobulin 8.52 � 103 1.35 � 10�3 6.30 � 106Endo H-treated thyroglobulin 1.04 � 104 1.36 � 10�3 7.68 � 106Fetuin 5.07 � 104 6.20 � 10�3 8.17 � 106Asialo-fetuin 1.26 � 104 6.04 � 10�3 2.09 � 106De-O-glycosylated fetuin 4.57 � 104 5.46 � 10�3 8.38 � 106De-N-glycosylated fetuin 9.71 � 10 4.67 � 10�3 2.08 � 104BSM 1.08 � 104

(B) BPTBovine thyroglobulin 1.55 � 104 1.45 � 10�6 1.07 � 1010�-Galactosidase-treated bovine thyroglobulin 3.19 � 10 9.07 � 10�5 3.51 � 105Porcine thyroglobulin 3.06 � 104 6.38 � 10�4 4.79 � 107Asialo-porcine thyroglobulin 4.28 � 103 6.08 � 10�4 7.04 � 106Endo H-treated thyroglobulin 3.49 � 102 5.33 � 10�5 6.55 � 106Fetuin 2.43 � 104 5.67 � 10�3 4.30 � 106Asialo-fetuin 9.13 � 103 7.13 � 10�3 1.28 � 106De-O-glycosylated fetuin 1.79 � 104 5.40 � 10�3 3.32 � 106De-N-glycosylated fetuin 1.09 � 102 1.86 � 10�2 5.87 � 103BSM 8.09 � 104




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DISCUSSION

This study demonstrates that mammalian pancreatic trypsin com-monly binds to glycoproteins possessing N-linked glycans by carbohy-drate-specific interaction. The sugar-binding specificity of trypsin wasshown by the binding with sugar-BP probes and glycolipid analogues tobe �-galactosyl, oligomannosyl, and nonreducing terminal �2,6-NeuAcresidues (Fig. 1). Trypsin bound to glycoproteins possessing N-glycanswith very high affinity, reaching 1010-106 M�1, whereas it did not bind toBSM (Fig. 4 and Table 1). The binding of glycoprotein probes withtrypsin was changed by glycosidase treatments on ELISA and SPR anal-yses, which coincided well with the sugar-binding specificity indicatedby sugar-BP probes. The specificity of the interaction between trypsinand the glycoproteins was proven by inhibition studies with monosac-

charides using SPR (Fig. 7) and conclusively demonstrated to be due tothe affinity of trypsin for component saccharide residues of theN-linkedglycans but not by protein-protein interaction.Treatment of trypsin with soybean trypsin inhibitor and PMSF did

not affect the binding to sugar-BP, glycolipid analogues, and glycopro-tein probes, and trypsin was noncompetitively and uncompetitivelyactivated toward synthetic substrates, BAEE and BAPA, by the bindingof specific sugars (Fig. 9 and Table 2). Therefore, theN-glycan recogni-tion of trypsinmust be exhibited at a site different from its catalytic site,and activation would be caused by an allosteric effect to make the sub-strate-binding site more accessible to the substrate and/or by a confor-mational effect that stabilizes the trypsin molecule against autodegra-dation, like the stabilizing effect of Ca2� binding (14).Thecoatingof oligosaccharidesonglycoproteins can serve toprotect the

polypeptide chain from degradation by proteases (3). The contributions ofsialylation to the stabilizationof glycoproteinagainst tryptichydrolysishavebeen reported for several glycoproteins, including orosomucoid (15) andvitronectin (16).Thede-N-glycosylationof ovomucoidwith trifluorometh-anesulfonic acid has been reported to interfere with the inhibitory activityagainst trypsin andmake ovomucoid easily hydrolyzable with trypsin (17).Although the relationship between oligosaccharide structure and the pro-tective function against proteases has been explored for several glycopro-teins (18–20), the protectingmechanism achieved by the oligosaccharideshas remained unclear. Because the removal of oligosaccharides from amature protein does not always drastically alter its sensitivity to proteolysis,

FIGURE 9. Effect of various sugars on enzymeactivity of PPT. A, aliquots (100 �l) of 0.2 M methyl-�-mannoside, methyl-�-galactoside, or lactose, orbuffer for the control were added to PPT (2 �g) inthe same volume, and the enzyme activity wasmeasured against BAEE as described in the text.Relative activity was expressed as a percentage,taking the control as 100%. B, time course of PPTactivity in the presence of various sugars. Theenzyme activity was measured against BAPA asdescribed in the text in the presence or absence of0.2 M methyl-�-mannoside (f), methyl-�-galacto-side (�), lactose (�), and control (�). C–E, Lin-eweaver-Burk plots of PPT. PPT activity was meas-ured in the presence (solid line) or absence (dashedline) of 0.2 M concentrations of various sugars asdescribed in the text and analyzed by a doublereciprocal Lineweaver-Burk plot.

TABLE 2Km value and Vmax of PPT activity on effect of various sugarsThe enzyme activity was measured in the presence of various sugars (0.2 M) andanalyzed by the Lineweaver-Burk plot.

Sugars or glycoprotein Vmax Km Mode of effect� 10�3 M/s � 10�3 M

Control 8.08 1.26Me-�-Man 20.7 3.20 Uncompetitive

activationMe-�-Gal 18.1 2.81 Uncompetitive

activationLac 4.70 0.85 Non- and uncompetitive

inhibition




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some specific interaction between protease and glycoproteins may beinvolved in regulating protease attack.We found that trypsin sugar-specif-ically interacts with N-linked glycoproteins. The binding of trypsin to theN-glycans of glycoprotein would protect the carrier glycoprotein fromhydrolysis, at least partially, by topologically restricting the substrate-bind-ing site of trypsin. Deglycosylation of glycoproteins, which diminishes thecarbohydrate-specific binding, makes trypsin interact with the peptidemoiety of the glycoprotein through the substrate-binding site to hydrolyzeit. In this hypothesis, glycosylation at even one site of the polypeptide cansignificantly affect the proteolysis of the carrier glycoprotein. It is necessaryto define the relationship between the structure and position of glycosyla-tion that affects the susceptibility to trypsin to examine the hypothesis.The binding specificity of trypsin toward carbohydrates was different

from that of PPA. Trypsin bound little to N-acetyllactosamine and�-GalNAc, which boundwell to PPA, and bound to fetuin better than totransferrin (Fig. 4), whereas PPA bound to transferrin better than tofetuin (6). The differences may suggest that the endogenous receptorsfor trypsin and �-amylase are not identical.Thecarbohydrate-bindingactivityof trypsinwas exhibitedat abroadpH

range with the optimum at pH 8.0, the slightly alkaline pH similar to thepancreatic fluid in the intestinal lumen. Therefore, trypsin may interactwith glycoligands in the epithelial surface of the duodenumand intestine invivo, because it is extensively glycosylatedwithN-glycans (21–23) contain-ing �-galactose residues (24). The N-glycan-binding activity would play arole in targeting trypsin and concentrating it on the brush-border mem-brane. Such immobilization of trypsin would enhance the activity and/orelongate the short life span of trypsin by stabilizing it against autodegrada-tion. Because the ability of the duodenum to digest proteins increases rap-idly in the cascade of enzyme activations following trypsin activation, theenhancement of trypsin activity would be amplified to increase digestiveefficiency exponentially; therefore, the 140% enhancement of trypsin activ-ity measured by using BAEE would be amplified in the duodenum toincrease more than 5-fold after the sixth stage. In addition, the binding oftrypsin to intestinal epitheliummakes the product peptides spatially avail-able as a substrate for the exo-typepeptidases that arenaturally anchored tothe intestinal brush-border membrane. The activated proteinases cooper-atively break down dietary proteins to peptides that are subsequentlydegraded to amino acids by other exo-type peptidases either secreted orexpressed in the brush border membrane of epithelial cells in the duode-numand small intestine. Rat aminopeptidaseN (EC3.4.11.2) is one of suchglycoproteins with 20% (w/w) carbohydrates that possess unsialylated tri-and tetraantennary complex types as majorN-glycans (25), which are verysimilar to the glycans of ovomucoid.Amajor part of dipeptidylpeptidase IV(EC 3.4.14.5) (26) and peptide transporter-1, a H�/peptide cotransporterresponsible for the uptake of small peptides (27), are among theN-glycosy-lated glycoproteins in the small intestine, too. The association of trypsinwith this exopeptidase or transporter would enhance the rate of degrada-tionof substrate proteins andpeptide absorptionby increasing the catalyticefficiency both allosterically and with mass action after. The binding oftrypsin to intestinal glycoreceptorsmay also stimulate exocrine secretionofdigestive tract hormones or pancreatic proteins as reported for exog-enously administered plant lectins (28). The carbohydrate binding mayregulate the reactivity of trypsin with the glycosylated protease-activatedreceptor2dependingon theglycosylation state (29) and influence intestinalinflammation, cytoprotection, and cellular motility.Together with our previous findings on pancreatic �-amylase, carbo-

hydrate-binding activities of macromolecule-degrading enzymes might

play essential roles in localization, activation, and stabilization of pan-creatic enzymes to achieve efficient digestion. Considering the biologi-cal significance of trypsin in the activation of other proteinases and itsdegradative role in various tissues, themechanismofmodulating trypticsusceptibility by glycosylation of proteins must be elucidated.

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Hiroko Takekawa, Chieko Ina, Reiko Sato, Kazunori Toma and Haruko Ogawaof Glycoproteins

-Linked GlycansNNovel Carbohydrate-binding Activity of Pancreatic Trypsins to

doi: 10.1074/jbc.M513773200 originally published online January 17, 20062006, 281:8528-8538.J. Biol. Chem.

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