THE JOURNAL OF BIOLOGICAL CHEM1STRY Vol. 257, No. 18, Issue of September 25, pp. 10776-10780, 1982 Printed in U.S.A. Oligosaccharide Accessibility to Peptide:N-glycosidase as Promoted by Protein-unfolding Reagents* (Received for publication, March 24, 1982) Anthony L. Tarentino and Thomas H. Plummer, Jr. From the Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201 The ability of almond emulsin peptide:N-glycosidase to remove oligosaccharide chains from intact glycopro- teins was studied. Protein conformation appeared to be the main factor affecting carbohydrate removal. In the native state the oligosaccharides of ribonuclease B and the Fab, fragment derived from immunoglobulin M were completely resistant to the enzyme, indicating that the polypeptide chain restricts access to the site of hydrolysis. Heat denaturation in sodium dodecyl sul- fate rendered these glycoproteins susceptible to pep- tide:N-glycosidase, but perturbation with chaotropic salts provided a more gentle approach, which was as effective as detergent-unfolding and more compatible with the stability of the enzyme. Once exposed by the unfolding reagents, the complex oligosaccharides of Fab, were released more rapidly than the high mannose chains of ribonuclease B, consistent with their prefer- ential release from small glycopeptides (Plummer, T. H., Jr., and Tarentino, A. L. (1981) J. BioL Chem. 256, 10243-10246). Peptide:N-glycosidase was first isolated from almond emul- sin by Takahashi (1) and Takahashi and Nishibe (2) and shown to hydrolyze the f8-aspartylglycosylamine linkage in a series of related ovalbumin and bromelain glycopeptides. Two major products were demonstrated from the hydrolysis of these N-linked glycopeptides: a carbohydrate-free peptide containing an aspartic acid residue, and an intact oligosaccha- ride with N-acetylglucosamine on the reducing end (2). Re- cently we purified the enzyme and examined its specificity with respect to oligosaccharide chain length, type, and posi- tion along the peptide backbone (3). Our studies established that the enzyme could release sialic acid-containing complex biantennary oligosaccharides, as well as high mannose and hybrid forms, and that the former were the preferred sub- strates. Even endo-,f-N-acetylglucosaminidase H-derived gly- copeptides containing N-acetylglucosamine instead of a com- plete oligosaccharide chain were hydrolyzed completely, albeit at a slower rate than the parent glycopeptide (3). Given its broad oligosaccharide specificity, peptide:N-gly- cosidase could be a useful tool for characterizing the biosyn- thetic and processing pathways of N-glycosylated proteins and for determining oligosaccharide structure (4). This en- zyme would complement the proven utility of endo-,B-N-ace- tylglucosaminidase H for such studies (5-10). However, re- * This work was supported in part by National Institutes of Health Grants GM23900 and GM30471 from the National Institute of General Medical Sciences, United States Public Health Service, Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. moval of oligosaccharides from intact glycoproteins is much slower than removal from glycopeptides (11), and thus far complete deglycosylation has not been achieved. For example, only 38% of the carbohydrate was removed from desialyated fibrinogen and its fi- and y-polypeptide chains, whereas over 98% of the oligosaccharides were released from the corre- sponding pepsin-derived glycopeptides (12). Incomplete re- lease of oligosaccharides was observed also from ovalbumin, bromelain, and desialyated transferrin (11), suggesting that access of peptide:N-glycosidase to the site of hydrolysis is restricted when glycoproteins are in their native state. In preliminary studies with ovalbumin, we also observed that all oligosaccharide chains could be removed easily from defined glycopeptides, but in the intact glycoprotein the chains were resistant to the enzyme. These findings prompted us to investigate more closely the question of oligosaccharide accessibility to peptide:N-glycosidase and to develop condi- tions which would make this enzyme more suitable for use with glycoproteins. In this report we show that chaotropic salts, which perturb water structure and weaken hydrophobic interactions (13), promote the deglycosylation of high man- nose and complex glycoproteins by peptide:N-glycosidase. EXPERIMENTAL PROCEDURES 1 ' 2 Enzymes-Peptide:N-glycosidase was partially purified (300-fold) from 10 g of crude almond emulsin (Sigma). Assays were conducted with ovalbumin [ 3 H]dns-octaglycopeptide at a specific radioactivity of 130 x 103 dpm/nmol. Reactions were conducted in 1.0-ml conical disposable plastic microtubes and contained 3 l of radioactive sub- strate and 3 d of appropriately diluted enzyme in 0.01 M sodium acetate, pH 5.1 (3). One unit of enzymatic activity represents 1 nmol of substrate hydrolyzed/min at 37 C. The enzyme was obtained in 26% yield with a specific activity of 804 nmol of [ 3 H]dns-octapeptide formed/min/mg of protein. The preparation was devoid of a-man- nosidase but contained traces of i-galactosidase and i-N-acetylglu- cosaminidase, which, as demonstrated previously (3) and again in this study, did not affect the experimental results. Glycoprotein Substrates-RNase B was purchased from Sigma and purified by affinity chromatography on ConA-Sepharose (14). IgM (Ga) was isolated from the plasma of a patient with Walden- strom's macroglobulinemia (15). The Fab, fragment containing the 'Portions of this paper (including part of "Experimental Proce- dures," Figures 1 and 2, Table I, and additional references) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-5148, cite the authors and include a check or money order for $2.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. 2 The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- polyacrylamide slab gel electrophoresis; CHO(+), carbohydrate-con- taining; CHO(-), carbohydrate-free; SDS, sodium dodecyl sulfate; RNase B, ribonuclease B; dns, 5-dimethylaminonaphthalene-l-sul- fonyl; ConA, concanavalin A; PMSF, phenylmethanesulfonyl fluoride. 10776 by guest on July 25, 2020 http://www.jbc.org/ Downloaded from
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THE JOURNAL OF BIOLOGICAL CHEM1STRYVol. 257, No. 18, Issue of September 25, pp. 10776-10780, 1982Printed in U.S.A.
Oligosaccharide Accessibility to Peptide:N-glycosidase as Promoted byProtein-unfolding Reagents*
(Received for publication, March 24, 1982)
Anthony L. Tarentino and Thomas H. Plummer, Jr.
From the Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201
The ability of almond emulsin peptide:N-glycosidaseto remove oligosaccharide chains from intact glycopro-teins was studied. Protein conformation appeared to bethe main factor affecting carbohydrate removal. In thenative state the oligosaccharides of ribonuclease B andthe Fab, fragment derived from immunoglobulin Mwere completely resistant to the enzyme, indicatingthat the polypeptide chain restricts access to the site ofhydrolysis. Heat denaturation in sodium dodecyl sul-fate rendered these glycoproteins susceptible to pep-tide:N-glycosidase, but perturbation with chaotropicsalts provided a more gentle approach, which was aseffective as detergent-unfolding and more compatiblewith the stability of the enzyme. Once exposed by theunfolding reagents, the complex oligosaccharides ofFab, were released more rapidly than the high mannosechains of ribonuclease B, consistent with their prefer-ential release from small glycopeptides (Plummer, T.H., Jr., and Tarentino, A. L. (1981) J. BioL Chem. 256,10243-10246).
Peptide:N-glycosidase was first isolated from almond emul-sin by Takahashi (1) and Takahashi and Nishibe (2) andshown to hydrolyze the f8-aspartylglycosylamine linkage in aseries of related ovalbumin and bromelain glycopeptides. Twomajor products were demonstrated from the hydrolysis ofthese N-linked glycopeptides: a carbohydrate-free peptidecontaining an aspartic acid residue, and an intact oligosaccha-ride with N-acetylglucosamine on the reducing end (2). Re-cently we purified the enzyme and examined its specificitywith respect to oligosaccharide chain length, type, and posi-tion along the peptide backbone (3). Our studies establishedthat the enzyme could release sialic acid-containing complexbiantennary oligosaccharides, as well as high mannose andhybrid forms, and that the former were the preferred sub-strates. Even endo-,f-N-acetylglucosaminidase H-derived gly-copeptides containing N-acetylglucosamine instead of a com-plete oligosaccharide chain were hydrolyzed completely, albeitat a slower rate than the parent glycopeptide (3).
Given its broad oligosaccharide specificity, peptide:N-gly-cosidase could be a useful tool for characterizing the biosyn-thetic and processing pathways of N-glycosylated proteinsand for determining oligosaccharide structure (4). This en-zyme would complement the proven utility of endo-,B-N-ace-tylglucosaminidase H for such studies (5-10). However, re-
* This work was supported in part by National Institutes of HealthGrants GM23900 and GM30471 from the National Institute of GeneralMedical Sciences, United States Public Health Service, Departmentof Health and Human Services. The costs of publication of this articlewere defrayed in part by the payment of page charges. This articlemust therefore be hereby marked "advertisement" in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
moval of oligosaccharides from intact glycoproteins is muchslower than removal from glycopeptides (11), and thus farcomplete deglycosylation has not been achieved. For example,only 38% of the carbohydrate was removed from desialyatedfibrinogen and its fi- and y-polypeptide chains, whereas over98% of the oligosaccharides were released from the corre-sponding pepsin-derived glycopeptides (12). Incomplete re-lease of oligosaccharides was observed also from ovalbumin,bromelain, and desialyated transferrin (11), suggesting thataccess of peptide:N-glycosidase to the site of hydrolysis isrestricted when glycoproteins are in their native state.
In preliminary studies with ovalbumin, we also observedthat all oligosaccharide chains could be removed easily fromdefined glycopeptides, but in the intact glycoprotein thechains were resistant to the enzyme. These findings promptedus to investigate more closely the question of oligosaccharideaccessibility to peptide:N-glycosidase and to develop condi-tions which would make this enzyme more suitable for usewith glycoproteins. In this report we show that chaotropicsalts, which perturb water structure and weaken hydrophobicinteractions (13), promote the deglycosylation of high man-nose and complex glycoproteins by peptide:N-glycosidase.
EXPERIMENTAL PROCEDURES 1' 2
Enzymes-Peptide:N-glycosidase was partially purified (300-fold)from 10 g of crude almond emulsin (Sigma). Assays were conductedwith ovalbumin [3 H]dns-octaglycopeptide at a specific radioactivityof 130 x 103 dpm/nmol. Reactions were conducted in 1.0-ml conicaldisposable plastic microtubes and contained 3 l of radioactive sub-strate and 3 d of appropriately diluted enzyme in 0.01 M sodiumacetate, pH 5.1 (3). One unit of enzymatic activity represents 1 nmolof substrate hydrolyzed/min at 37 C. The enzyme was obtained in26% yield with a specific activity of 804 nmol of [3H]dns-octapeptideformed/min/mg of protein. The preparation was devoid of a-man-nosidase but contained traces of i-galactosidase and i-N-acetylglu-cosaminidase, which, as demonstrated previously (3) and again in thisstudy, did not affect the experimental results.
Glycoprotein Substrates-RNase B was purchased from Sigmaand purified by affinity chromatography on ConA-Sepharose (14).IgM (Ga) was isolated from the plasma of a patient with Walden-strom's macroglobulinemia (15). The Fab, fragment containing the
'Portions of this paper (including part of "Experimental Proce-dures," Figures 1 and 2, Table I, and additional references) arepresented in miniprint at the end of this paper. Miniprint is easilyread with the aid of a standard magnifying glass. Full size photocopiesare available from the Journal of Biological Chemistry, 9650 RockvillePike, Bethesda, MD 20814. Request Document No. 82M-5148, citethe authors and include a check or money order for $2.40 per set ofphotocopies. Full size photocopies are also included in the microfilmedition of the Journal that is available from Waverly Press.
2 The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis; CHO(+), carbohydrate-con-taining; CHO(-), carbohydrate-free; SDS, sodium dodecyl sulfate;RNase B, ribonuclease B; dns, 5-dimethylaminonaphthalene-l-sul-fonyl; ConA, concanavalin A; PMSF, phenylmethanesulfonyl fluoride.
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Carbohydrate Removal from Glycoproteins
The stability properties of peptide:N-glycosidase (Fig. 3B)afford a wide latitude in selecting appropriate conditions. Forexample, carbohydrate depletion of CHO(+)Fab, in 0.5 MNaSCN at 37 °C proceeded rapidly for 6 h, then leveled off atabout 89% completion (Fig. 3A, O-O). Increasing or de-creasing the NaSCN concentration or including thiols did notresult in further carbohydrate release. However, in 0.5 MNaSCN at 46 C the enzyme was stable sufficiently long (Fig.3B) to allow for a more rapid and complete (>97%) deglyco-sylation (Fig. 3A, A A). Peptide:N-glycosidase is com-pletely stable in 0.75 M NaSCN for 48 h at 37 C (Fig. 3B),and in 1.0 to 1.5 M NaSCN a 50% activation is routinelyobserved (data not shown).
At these high salt concentrations many glycoproteins shouldbe in an unfolded state and thus more susceptible to pep-tide:N-glycosidase. In preliminary studies with ovomucoid,which contains about 25% carbohydrate by weight (29), >80%of the N-glycosylated chains were released in a reaction with1.0 M NaSCN at 37 C.
Peptide:N-glycosidase has great potential for use in struc-tural analysis of glycopeptides, and addition of chaotropicsalts and/or detergents may extend its utility to glycoproteinsas well. Modification of protein structure by alkylating agents,detergents, and thiols has been successfully employed to pro-mote deglycosylation of high mannose glycoproteins by endo-,-N-acetylglucosaminidase H (5, 6, 19, 30). Use of peptide:N-glycosidase with chaotropic reagents provides a mild alterna-tive for the release of oligosaccharide chains and for studyingthe influence of carbohydrate on protein conformation.
Acknowledgments-We express our appreciation to Patricia Fox,Matthew T. Kimmel, and Arthur W. Phelan for excellent technicalassistance and to Dr. Samuel Murphy for a generous supply of IgM.
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A L Tarentino and T H Plummer Jrreagents.
Oligosaccharide accessibility to peptide:N-glycosidase as promoted by protein-unfolding
1982, 257:10776-10780.J. Biol. Chem.
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