Molecular Cloning, Primary Structure, and Properties of a New Glycoamidase from the Fungus Aspergillus tubigensis*

A new glycoamidase, peptide-N 4-(N-acetyl-β-d-glucosaminyl)asparagine amidase (PNGase) At, was discovered in the eukaryote Aspergillus tubigensis. The enzyme was purified to homogeneity, and the DNA sequence was determined by cloning in Escherichia coli. Over 80% of the deduced amino acid sequence was verified independently by Edman analysis and/or electrospray ionization-mass spectrometry of protease fragments of native PNGase At. This glycoamidase contains 12 potential asparagine-linked glycosylation sites, of which at least 9 sites are occupied with typical high mannose oligosaccharides. PNGase At consists of two non-identical glycosylated subunits that are derived from a single polypeptide gene precursor. Evidence is presented suggesting that autocatalysis is involved in subunit formation. PNGase At is an important new tool for analysis of asparagine-linked glycans; it can hydrolyze a broad range of glycopeptides, including those with core-linked α1→6 or α1→3 fucose, under conditions not favorable with existing glycoamidases.

Glycoamidases are an important class of deglycosylating enzymes, and their use has contributed greatly to our knowledge of the structure/function of asparagine-linked glycoproteins. Peptide-N 4 -(N-acetyl-␤-D-glucosaminyl)asparaginase amidase F (PNGase F) 1 is an amidase/amidohydrolase that cleaves the ␤-aspartylglucosylamine bond of peptide-bound asparaginelinked glycans, converting the asparagine residue to aspartic acid and releasing an intact 1-amino oligosaccharide (1). This enzyme has been purified to homogeneity from the Gramnegative bacterium Flavobacterium meningosepticum, and its substrate specificity has been well established (2). PNGase F has been cloned and sequenced (3), and its crystallographic structure and active center were determined to 2.2 Å (4,5). Other glycoamidases catalyzing the same reaction have been reported in plants (e.g. almond PNGase A) (6,7) and, more recently, in mammalian cells (8), where the enzyme is thought to have a regulatory role in glycoprotein maturation (9). However, only the bacterial glycoamidase PNGase F has been characterized at the molecular level, and an understanding of the primary structure of even the simplest eukaryotic glycoamidase is still lacking.
During the course of a glycoamidase survey, we discovered high levels of a new PNGase type enzyme in crude commercial pectinase extracts derived from Aspergillus tubigensis. Preliminary studies demonstrated that this glycoamidase, designated PNGase At, appeared significantly different from PNGase F in terms of its molecular weight, state of glycosylation, low pH optimum, and thermostability as to warrant further investigation into its protein structure and mechanism of action. In this report we describe the purification of PNGase At to homogeneity. The enzyme has been cloned and sequenced, and the deduced amino acid sequence verified independently to greater than 80% by ESI-MS. On the basis of the structural organization of the gene, PNGase At is initially synthesized as a single polypeptide of about 59,000 daltons. It acquires asparaginelinked glycans during post-translational processing and is cleaved into non-identical subunits. Subunit formation represents a fundamental departure from the structure of the corresponding bacterial enzyme and may reflect a different mechanism for catalysis of the ␤-aspartylglucosylamine bond. This study represents the first description of a glycoamidase at the eukaryotic level.

Materials
A. tubigensis strain 7 DNA, RNA, and Multifect PL enzyme were obtained from Genencor. Pfu polymerase, Bluescript KSϩ, SKϩ, and PCR script SKϩ cloning vectors were obtained from Stratagene. Reverse transcriptase, RNase inhibitor, and RNase-free DNase were from Promega. T4 DNA ligase, polynucleotide kinase, and all the restriction enzymes used in this project were from New England Biolabs.

Polymerase Chain Reactions
PCR amplification reactions were performed using 100 ng of A. tubigensis total DNA, 100 pmol of each primer, 200 mM dNTP, 2.5 units Pfu DNA polymerase, and 1 ϫ Pfu buffer (Stratagene) in a total volume of 100 l. The reactions were carried out in a DNA thermal cycler (Perkin-Elmer) programmed for 30 three-step cycles of denaturation (1 min at 94°C), annealing (1 min at either 45°C for degenerate primers or 55°C for specific primers), and elongation (1 min at 70°C). A further elongation at 72°C was performed for 10 min at the end of the program.

Molecular Cloning of PNGase At
Based on the peptide sequence analysis, two oppositely oriented degenerated oligonucleotides were designed, AnS1 (5Ј-ACAGAATTC-GARGTNTTYGARGTNTAYCARCC-3Ј, amino acid residues 3-10) and AnA4 (5Ј-ACAAAGCTTCGRAANACYTCNGTRTCNCC-3Ј, amino acid residues 75-81) where N is either G, A, T, or C, R is G or A, and Y is C * This work was supported in part by Grant 30471 from the NIGMS, United States Public Health Service, DHHS (to A. L. T. and T. H. P.). 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) U96923.
‡ To whom correspondence should be addressed. Tel.: 518-486-2578; Fax: 518-473-2900; E-mail: ftouhi@wadsworth.org. 1 The abbreviations used are: PNGase, peptide-N 4 -(N-acetyl-␤-D-glucosaminyl)asparagine amidase; Endo, endo-␤-N-glucosaminidase; ESI-MS, electrospray ionization-mass spectrometry; MS, mass spectrometry; PCR, polymerase chain reaction; bp, base pair(s); RAGE, rapid amplification of genomic DNA ends; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Dns, 5-dimethylaminonaphthalene-1-sulfonyl. or T. Using these primers, a PCR product of 294 bp was obtained. This fragment was digested with EcoRI and HindIII restriction enzymes and cloned into pBlueScript (Stratagene) pre-digested with the same enzymes. The sequence reactions were performed using a Sequenase kit (U. S. Biochemical Corp.). To extend the sequence further, upstream and downstream regions of this fragment were amplified using RAGE (10) and rapid amplification of cDNA ends (11) protocols. Briefly, in vitro polyadenylated cDNAs (rapid amplification of cDNA ends) or restriction fragments of genomic DNA (RAGE) were used in PCR reactions with a specific primer and an oligo(dT) to amplify unknown sequences. Using this information, the whole gene was then amplified in two large fragments. For amplifying fragment A, the following primers were used, AnA17 (5Ј-ATCTCGAACGAATGGGACTTGTTG-3Ј, nucleotides 912-935) and AnS18 (5Ј-GAAATCTCCGCATGCAGAG-CACGT-3Ј (5Ј-translated region, not shown)). A second, nested PCR amplification was performed using primers AnA8 (5Ј-ATACTCATCG-GCAGAGAGGACGTTG-3Ј, nucleotides 669 -693) and AnS18, resulting in a fragment of 1075 bp long. The same strategy was applied to amplify fragment B as follows: the first PCR reaction was carried out with primers AnS11 (5Ј-CAACGCCACAGTGCAATATCAGATC-3Ј, nucleotides 573-597) and AnA19 (5Ј-CTAGCTCTCAGTATCCGAAACAAC-CGT-3Ј, nucleotides 1651-1677). A nested PCR reaction was performed using primers AnS6 (5Ј-ACAGCATCCCGTGCAGTCGTGTCC-3Ј, nucleotides 604 -627) and AnA19. The PCR end product was 1074 bp long. Fragments A and B were cloned into PCRScript (Stratagene), and the nucleotide sequence for each of them was determined from two independent clones. The reconstruction of the gene from these two fragments was done in Bluescript SKϩ.

Enzyme Purification
All operations were performed at room temperature. Sephadex G-75-150 ml of the crude dark brown Multifect PL extract (Genencor) was applied to a 7.6 ϫ 58-cm column of Sephadex G-75 (63-88 M) equilibrated in 10 mM sodium acetate, pH 5.2, containing 1% butanol. The column was developed at 190 ml/h, and 19-ml fractions were collected. PNGase At eluted sharply after the void volume (tubes 42-64) and was well separated from most of the dark brown pigmented material.
Toyopearl DEAE 650S-The PNGase At Pool from two gel filtration runs was applied to a 2.0 ϫ 11-cm column of Toyopearl DEAE 650 (35 M) equilibrated in 20 mM sodium acetate, pH 5.2. A linear gradient was developed from 0 to 0.4 M NaCl over 4 h with a Waters 650 E advanced protein purification system. The flow rate was 300 ml/h, and 10-ml fractions were collected. PNGase At fractions were pooled from tubes 31 to 68 (0.05-0.15 M NaCl).
Hydrophobic Interaction Chromatography-Pooled fractions were diluted 10% by volume of 0.5 M sodium phosphate, pH 7.0, brought to pH 7.3 with cold 1 N NaOH, and adjusted to 1 M ammonium sulfate. The extract was applied to a 1.6 ϫ 47-cm column of Toyopearl butyl-650 M equilibrated in 100 mM sodium phosphate, pH 7.0, containing 1 M ammonium sulfate and 10 mM EDTA. A deceasing linear gradient (Waters, curve 6) to 50 mM sodium phosphate, 10 mM EDTA was generated at 120 ml/h over 7 h, and 5-ml fractions were collected. The enzyme began eluting from the column at about 0.5 M ammonium sulfate.
Protein-Pak DEAE-Pooled fractions from the previous step were dialyzed against 20 mM sodium acetate, pH 5.2, and applied to a 1.6 ϫ 11 cm column of Protein-Pak DEAE 15 HR (15 M, Waters). A linear gradient was developed from 0 to 0.3 M NaCl over 3.5 h at a flow of 180 ml/h, and 4.5-ml fractions were collected.
Lectin Affinity Chromatography-The PNGase At fractions from above were pooled and concentrated by ultrafiltration (Amicon) to 19.5 ml, and the pH was raised to 7.5 with 1 M Tris chloride, pH 8.6. The sample was applied to a 0.9 ϫ 19-cm column of concanavalin A-Sepharose (Sigma, type VA) equilibrated in 10 mM Tris-Cl, pH 7.5, 300 mM NaCl, 1 mM CaCl 2 , and 1 mM MgCl 2 . The column was washed in starting buffer for 1 h at a flow of 24 ml/h and then developed with a linear gradient to 250 mM methyl-D-mannoside over 6.8 h. Fractions of 1.6 ml were collected. PNGase At eluted as the most retarded material in tubes 45-76.
Hydroxyapatite Chromatography-The pooled fractions were dialyzed against 10 mM sodium phosphate, pH 7.0, and the retentate was loaded on a 0.9 ϫ 14-cm column of Macro-Prep ceramic hydroxyapatite (type 1, Bio-Rad) equilibrated in 10 mM sodium phosphate, pH 7.0. The column was washed for 15 min at a flow rate of 30 ml/h and developed with a linear gradient from 0 to 0.1 M NaCl for 130 min.

Protease Digestions
PNGase At in its native state was digested with trypsin, endoproteinase Glu-C, or endoproteinase Asp-N in ammonium bicarbonate, pH 8, according to the manufacturer's recommendations (Boehringer Mannheim). Trypsin-insoluble peptides were further degraded with pepsin in 0.1% formic acid. Peptides were isolated from these digests by reverse-phase HPLC on 1 ϫ 150-cm Vydac columns. Unfractionated digests or purified glycopeptides were treated with PNGase F at pH 8.0 to release asparagine-linked oligosaccharides.

Mass Spectrometry
All spectra were acquired on a Finnigan TSQ 700 equipped with a Finnigan ESI source. Samples were introduced into the mass spectrometer by infusion at a flow rate of 0.3 l/min through a 20-m inner diameter fused silica capillary. Solvents were various mixtures of water/methanol/acetonitrile with 0.1% formic acid or 0.04% trifluoroacetic acid. LC-MS was accomplished with a homemade ϳ50:1 splitter to introduce sample into the mass spectrometer at 0.5 to 1 l/min. Peptide fractions from LC-MS were automatically collected for sequence analysis. Methyl ester derivatives were prepared by exposure to 1 N methanolic HCl. MS/MS spectra were generated at 20 -40 eV collision energy with argon as the collision gas.

Edman Microsequence Analysis
Automated Edman degradation was performed with a model 477A Applied Biosystem pulsed liquid sequenator equipped with a model 120A amino acid analyzer. Sequence analysis (50 -200 pmol) was conducted on samples isolated by HPLC or from Western blots on polyvinylidene difluoride membranes following SDS-PAGE (13).

Deglycosylation of PNGase At with Endo F 1
Native PNGase At (3.13 mg, 53 nmol) and equimolar Endo F 1 (1.77 mg, 56 nmol) were incubated at 37°C in 50 mM ammonium acetate, pH 6.0, in a total volume of 15.3 ml. At 20 h the reaction was terminated by lyophilization. The sample was redissolved in 10 ml of water, and the pH was adjusted to 5.2 with 0.1 N acetic acid. The conductivity of the solution was equalized to 20 mM sodium acetate, pH 5.2 by addition of water. To separate Endo F 1 from deglycosylated PNGase At, the diluted sample was applied to a 0.5 ϫ 10.5-cm column of Protein-Pak DEAE 15 HR (Waters) in 20 mM sodium acetate, pH 5.2, at a flow rate of 30 ml/h. The column was washed in starting buffer for 20 min to completely remove Endo F 1 , followed by a gradient from 0 to 0.25 M NaCl over 30 min. Deglycosylated PNGase At was eluted in a stepwise manner with 0.5 M sodium acetate, pH 5.2, and then 0.5 M sodium acetate, pH 6.5. Carbohydrate analysis of deglycosylated PNGase At indicated a very low hexose content, consistent with essentially complete removal of the asparagine-linked oligosaccharides.

Carbohydrate Analysis
Total carbohydrate was determined on PNGase At samples by the phenol-sulfuric acid method (14). Monosaccharide and glucosamine analyses were done using 2 N trifluoroacetic acid at 100°C for 5 h, and 4 N HCl at 100°C for 6 h, respectively (15). Parallel analyses were done using 2 N HCl at 100°for 2.5 h (16).

Preparation of Glycopeptides
Hen ovomucoid, GTN(CHO)ISK, was isolated from reduced carboxymethylated ovomucoid after tryptic followed by chymotryptic digestion. Glycopeptides were purified on Sephadex G-50 followed by chromatography on Dowex 50-X2 as described for IgM glycopeptides (17). Other glycopeptides were purified as indicated: bovine fetuin, LAN(CHO)AeCS (where AeC is aminoethylcysteine) and LAN(CHO)C-mCS (where CmC is carboxymethylcysteine) (18); porcine fibrinogen, VEN(CHO)K, and VGEN(CHO)R (19). Pineapple bromelein glycopeptide was isolated from a thermolytic digest of reduced carboxymethylated enzyme after chromatography on Sephadex G-50 (17) and Bakerbond Wide Pore C 18 (19). A modified glycopeptide containing a core GlcNAc with an ␣136-linked fucose was produced as follows: Dns VGEN(CHO)R (0.78 mol) was digested with Endo F 3 (15 g) in 400 l of 100 mM sodium acetate, pH 4.75, for 3 h at 37°C. The digestion mixture was fractionated on a 0.9 ϫ 130-cm column of Sephadex G-15 developed in 0.1 N acetic acid. The truncated glycopeptide eluting at 45-57 ml was the major peak and was lyophilized and quantitated for use in rate determinations. Glycopeptides were dansylated by the method of Tapuchi et al. (20).

Enzymes Assays
PNGase assays were performed by HPLC as described previously for Endo F 2 and Endo F 3 (2) but were done in 0.1 M sodium acetate, pH 5.2, for PNGase At and PNGase A and in 0.1 M HEPES, pH 8.8, for PNGase F. One milliunit corresponds to 1 nmol of glycopeptide hydrolyzed per min at 37°C at the appropriate pH.
Products from PNGase At-hydrolyzed glycopeptides were identified on HPLC by reference to the known cleavage products from PNGase A and PNGase F.

RESULTS AND DISCUSSION
PNGase Purification-Multifect PL enzyme is a highly concentrated commercial extract of the secretory enzymes from a proprietary strain (Genencor) of the fungus, A. tubigensis. It contains numerous glycosidases, carbohydrases, and proteases, etc. and is widely used in the food industry. The purification of PNGase At to homogeneity from this crude material is summarized in Table I. Typically, 300 ml of multifect PL extract yielded 4 mg of pure PNGase At at an average final specific activity of 53,860 nmol of di-dansyl fetuin glycopeptide hydrolyzed per min per mg. The enzyme migrated on SDS-PAGE as a heterogeneous band with an apparent M r of about 43,000 (Fig. 1, lane 1). Peptide fragments derived from purified PNGase At were used to design oligonucleotides for cloning the PNGase At gene.
Molecular Cloning and DNA Sequence Analysis-The PNGase At gene was cloned and sequenced in two fragments using PCR-based methodology. The gene was reconstructed as depicted in Fig. 2, and the complete sequence reconfirmed. The complete nucleotide sequence and the deduced amino acid sequence of the PNGase At gene is presented in Fig. 3. Edman analysis identified the amino terminus of native PNGase At as LLEVFEVYQ. . . . , but a background sequence was also present in the data and identified as another subunit only in retrospect (see below). Assuming that the first methionine at position Ϫ21 is the translation start codon, as is the case with most fungal genes (21), then the open reading frame of the gene encodes a typical hydrophobic signal peptide of 21 amino acids as expected for a protein secreted by A. tubigensis. The open reading frame contains a single 58-bp intron (Fig. 2) with the expected 5Ј-donor and 3Ј-acceptor splice sites (22) and codes for a mature protein of 537 amino acids with a predicted molecular average mass of 59,335 Da. Twelve potential asparagine-linked glycosylation sites (Asn-X-Ser/Thr) were identified and are highlighted in Fig. 3.
Peptide and Glycan Mapping-As shown in Table II, over 80% of the deduced amino acid sequence was corroborated independently by ESI-MS and Edman analysis of protease fragments of PNGase At. Of the 12 potential asparagine-linked glycosylation sites (Table III), sites 1, 6, and 11 were unoccupied and sites 9 and 12 were not identified at the peptide level. Sites 9 and 12 must be glycosylated since glucosamine and mannose analysis of the intact protein by phenol-sulfuric assay estimated a total of 9.6 oligosaccharide chains. Seven sites were shown by ESI-MS analysis to be fully or partially occupied with high mannose oligosaccharides. Man 6 GlcNAc 2 was found almost exclusively at site 2. ESI-MS analysis of permethylated Endo F1-released oligosaccharides as well as Dionex PA-100 chromatographic analysis demonstrated that all other glycosylation sites were predominantly Man 5 GlcNAc (71%), with small amounts of Man 6 -9 GlcNAc. At sites 3-5, 7, and 10, peptides were identified by MS analysis with a mass consistent with the presence of a single GlcNAc residue (5-10%). This finding suggests the action of a trace endoglycosidase in the Multifect PL preparation that cleaves the di-N-acetylchitobiose linkage at accessible sites in PNGase At, but thus far such an activity has not been detected.
One O-linked glycosylation site at Thr 352 was found by mass spectrometry; it contained mainly a single hexose (95%) and a small amount of a di-hexose (5%).
Subunit Structure-From the DNA sequence (Fig. 3) it is clear that the primary translation product of PNGase At is a single polypeptide with a predicted molecular mass of 59,335 Da. Post-transcriptional processing adds between 7 and 9 high mannose chains (average, Man 5 GlcNAc 2 ) increasing the mass by about 9 -11 kDa to over 70,000 Da. Because of the wellknown anomalous behavior of large glycoproteins during SDS-PAGE, it was expected that PNGase At would migrate with an apparent M r greater than its calculated mass of 70,000 daltons. However, six different preparations showed the same unusual result; PNGase At migrated on SDS-PAGE as a heterogeneous band with an apparent M r slightly less than 43,000 (Fig. 1, lane  1). This result suggests that PNGase At consists of two glycosylated subunits that dissociate in SDS and co-migrate on SDS-PAGE with an apparent M r around 43,000. After removal a The absorbance at 280 nm was used as an approximation to the protein concentration during purification. A 1% solution of pure PNGase At corresponds to an A 280 nm of 1.66 based on its amino acid composition (12). of the asparagine-linked oligosaccharides with Endo F 1 , deglycosylated PNGase At migrated as two well-resolved polypeptides (Fig. 1, lanes 2 and 4), an ␣-subunit with an apparent M r about 38,000 and a lighter staining ␤-subunit with an apparent M r of 28,000.
Additional evidence that PNGase At is composed of two non-identical subunits was provided by size-exclusion HPLC using the LKB Blue column. Under non-denaturing conditions PNGase At eluted in a high molecular weight form estimated to be 80,000 (Fig. 4A). However, after denaturation with SDS, the enzyme eluted as two subunits with an estimated M r of 42,000 and 26,000, respectively (Fig. 4B). Both the glycosylated and deglycosylated form of PNGase At were resolved into their subunits by HPLC, whereas on SDS-PAGE only the deglycosylated form was resolved. Note that A and B are not directly comparable by size versus elution time because SDS shifts the molecular weight standard curve to the left.
The amino acid sequence of the two subunits was determined on deglycosylated PNGase At by SDS-PAGE and Western blotting of the subunits to polyvinylidene difluoride membranes. Edman analysis (Table II)    NGT 528-530 ND a Asparagine-linked glycosylation sites determined from DNA sequence.
b Glycopeptides derived from trypsin, pepsin, or endo-Asp N digestions were analyzed by Edman or MS to determine the amino acid sequence (see Fig. 2), and by MS/MS for the carbohydrate at each site. Glycoforms are presented as 0, none present; or as oligosaccharides containing mannose (M) and N-acetylglucosamine (N) at the indicated composition. Single N-acetylglucosamine "stubs" are indicated by N.
c Not determined.
␤-subunits are present in approximately 1:1 ratio. Subunit formation occurs in a Ser/Thr-rich hydrophilic region of PNGase At between residues Thr 335 -Thr 336 (Fig. 3, **). At this point it has not been established whether this cleavage is the result of a specific protease or is caused by an autoproteolytic reaction. As a working hypothesis we favor the latter, because of the similarity of PNGase At to a new class of amidase/amidohydrolase enzymes, designated Ntn-amidohydrolases (reviewed in Ref. 23). Ntn-amidohydrolases (e.g. glycosylasparaginase) are derived from a single inactive precursor gene product via an autoproteolytic reaction. This mechanism places a reactive Thr, Ser, or Cys residue on the amino end of the ␤-subunit, where it functions as a combined base-nucleophile catalyst. If autoproteolysis is responsible for the Thr 336 form on the ␤-subunit of PNGase At, the enzyme should be only 40% active because 60% of the molecules have been degraded to the Ala 339 form. This conversion did not result from deglycosylation by Endo F 1 because native and deglycosylated PNGase At have the same specific activity. It is probably due to an aminopeptidase in the multifect PL extract.
Properties of PNGase At-PNGase At has no structural homology to PNGase F or to any other protein in the data bases. It has a 2-fold excess of acidic amino acids and a relatively low isoelectric point of 4.1. The pH activity curve is broad with a pH optimum at 5.0; at pH 3 and pH 8.6 the enzyme is 80 and 65%, respectively, of the maximum. In contrast PNGase F is less than 20% active at pH 5, and its pH optimum is between 8.6 and 9.0. Like many proteins secreted by A. tubigensis, PNGase At is relatively thermostable. It retains 65-70% of its original activity during incubation at 62°C for 8 h, unlike PNGase F, which is inactive after 1 h at 62°C. Glycopeptides can be hydrolyzed by PNGase At at 62°C at over three times the rate at 37°C. This is a useful property especially for glycopeptides with amino-terminal or carboxyl-terminal oligosaccharides, which are, in general, poor substrates for PNGase-type enzymes. Thus PNGase At is functional under conditions beyond the useful range for PNGase F.
A comparative analysis of the rates of hydrolysis of PNGase At and PNGase F toward selected glycopeptides is shown in Table IV. PNGase At has a broad substrate specificity hydrolyzing triantennary, biantennary, hybrid (Table IV), as well as high mannose-type (not shown) glycans. Triantennary glycans were preferred substrates for PNGase At and PNGase F. The negative charge of the carboxy-methyl group adjacent to the deglycosylation site adversely affects both enzymes compared with the corresponding amino-ethyl derivative. In general, the rates of hydrolysis by PNGase At were 3-4-fold slower at 37°C than for PNGase F, except for the last two substrates in Table  IV. As indicated in the preceding section, however, PNGase At may only be 40% active because of the loss of Thr 336 from the ␤-subunit. Interestingly, a truncated porcine fibrinogen glycopeptide (core fuc␣136GlcNAc) was hydrolyzed completely by PNGase At, albeit at a low rate, whereas it was completely resistant to PNGase F. This is an expected finding since a di-N-acetylchitobiose moiety is the minimum carbohydrate length required for PNGase F activity. Another indication of a difference in the active center of PNGase At and PNGase F is illustrated by the bromelain glycopeptide data in Table IV. PNGase At cleaved this glycopeptide at a very good rate, but it was totally resistant to PNGase F. The di-N-acetylchitobiosebinding domain in the active center of PNGase F allows for hydrolysis of glycans with fucose attached to the ␣136 position of the core proximal GlcNAc residue but lacks space for binding glycans with fucose in the ␣133 position (5).
PNGase At is more closely related in terms of substrate specificity to the almond enzyme PNGase A (glycopeptidase A) FIG. 4. Size exclusion HPLC of PN-Gase At under native conditions or after denaturation with SDS. The LKB column (7.5 ϫ 600 mm) was developed at 0.5 ml/min in 0.1 M sodium phosphate, pH 6.34 (native), or in the same buffer containing 0.1% SDS (denatured). A, native PNGase At; B, deglycosylated PNGase At preincubated in 1% SDS for 30 min at 60°C. Standard curves of molecular weight markers were done either native or denatured with SDS and used to estimate molecular weights of PNGase At samples. than to the bacterial enzyme PNGase F. PNGase At and PN-Gase A (24) both hydrolyze a broad array of oligosaccharide types, including very short glycans, as well as oligosaccharides with ␣136 or ␣133 core-linked fucose (25,26). PNGase At, like PNGase A, does not hydrolyze intact glycoproteins efficiently but prefers glycopeptide substrates. For two enzymes performing the same catalytic function, it is striking that PNGase At and PNGase F are so dissimilar; they have no primary structural homology, their molecular weight and subunit structure are very different, and their physical properties and specificity are different. Among the large endoglycosidase family of enzymes, for example, one always sees clear indications of relatedness in structure and function, but the glycoamidases PNGase F and PNGase At appear to have developed along different evolutionary lines. What path a mammalian enzyme would follow is unclear at this time.
We are currently developing a high expression system to study PNGase At. Unresolved questions will require a demonstration of the putative primary translation product and its conversion to ␣and ␤-subunits. X-ray crystallographic analysis of PNGase At and comparison with PNGase F will be important for investigating the development of the active center of these glycoamidases.