Site-directed Mutagenesis Study of Yeast Peptide: N-Glycanase

Yeast peptide:N-glycanase (Png1p; PNGase), a deglycosylation enzyme involved in the proteasome dependent degradation of proteins, has been reported to be a member of the transglutaminase superfamily based on sequence alignment. In this study we have investigated the structure-function relationship of Png1p by site-directed mutagenesis. Cys-191, His-218, and Asp-235 of Png1p are conserved in the sequence of factor XIIIa, where these amino acids constitute a catalytic triad. Point mutations of these residues in Png1p resulted in complete loss in activity, consistent with a role for each in catalyzing deglycosylation of glycoproteins. Other conserved amino acid residues, Trp-220, Trp-231, Arg-210, and Glu-222, were also vitally important for folding and structure stability of the enzyme as revealed by circular dichroism analysis. The potential effects of the mutations were predicted by mapping the conserved amino acids of Png1p within the known three-dimensional structure of factor XIIIa. Our data suggest that the lack in enzyme activity when any of the catalytic triad residues is mutated is either due to the absence of charge relay in the case of the triad or due to the disruption of the native fold of the enzyme. These findings strongly suggest a common evolutionary lineage for the PNGases and transglutaminases. Yeast peptide:N-glycanase (Png1p; PNGase), a deglycosylation enzyme involved in the proteasome dependent degradation of proteins, has been reported to be a member of the transglutaminase superfamily based on sequence alignment. In this study we have investigated the structure-function relationship of Png1p by site-directed mutagenesis. Cys-191, His-218, and Asp-235 of Png1p are conserved in the sequence of factor XIIIa, where these amino acids constitute a catalytic triad. Point mutations of these residues in Png1p resulted in complete loss in activity, consistent with a role for each in catalyzing deglycosylation of glycoproteins. Other conserved amino acid residues, Trp-220, Trp-231, Arg-210, and Glu-222, were also vitally important for folding and structure stability of the enzyme as revealed by circular dichroism analysis. The potential effects of the mutations were predicted by mapping the conserved amino acids of Png1p within the known three-dimensional structure of factor XIIIa. Our data suggest that the lack in enzyme activity when any of the catalytic triad residues is mutated is either due to the absence of charge relay in the case of the triad or due to the disruption of the native fold of the enzyme. These findings strongly suggest a common evolutionary lineage for the PNGases and transglutaminases. endoplasmic reticulum peptide:N-glycanase yeast PNGase dithiothreitol β-mercaptoethanol circular dichroism wild type 4-morpholineethanesulfonic acid Protein synthesis and folding is an essential process in eukaryotes that is accomplished for membrane and secretory proteins in the endoplasmic reticulum (ER).1 The fate of the newly synthesized protein, whether it is destined for secretion or degradation, is based on its conformation. Proteins with a native conformation enter Golgi transport vesicles and are then sent to other destinations. However, misfolded or non-native proteins cannot enter Golgi transport vesicle and are retained in ER (1.Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (589) Google Scholar). In some cases these misfolded proteins are subjected to degradation by ER-associated degradation mechanism (2.Kopito R.R. Cell. 1997; 88: 427-430Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 3.Cresswell P. Hughes E.A. Curr. Biol. 1997; 7: R552-R555Abstract Full Text Full Text PDF PubMed Google Scholar, 4.Suzuki T. Yan Q. Lennarz W.J. J. Biol. Chem. 1998; 273: 10083-10086Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 5.Plemper R.K. Wolf D.H. Trends Biochem. Sci. 1999; 24: 266-270Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 6.Brodsky J.L. McCracken A.A. Semin. Cell Dev. Biol. 1999; 10: 507-513Crossref PubMed Scopus (300) Google Scholar), in which misfolded protein is transferred from the ER to the cytosol, followed by degradation by the proteasome. In the case of misfolded glycoproteins that are transferred from the lumen of ER to the cytosol for degradation, the cleavage of the N-linked glycan is catalyzed by the enzyme peptide:N-glycanase (PNGase), which hydrolyzes the β-aspartylglycosylamine bond of aspargine-linked glycopeptides and glycoproteins and may act in concert with the proteasomal degradation of proteins that are exported out of the ER to the cytosol (7.Wiertz E.J.H.J. Jones T.R. Sun L. Bogyo M. Geuze H.J. Ploegh H.L. Cell. 1996; 84: 769-779Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, 8.Wiertz E.J.H.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (955) Google Scholar). The gene PNG1 encoding for the cytoplasmic deglycosylating enzyme has been identified in yeast and was found to be well conserved throughout the eukaryotes (9.Suzuki T. Park H. Hollingsworth N. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1052Crossref PubMed Scopus (190) Google Scholar). More recently, physical interaction between yeast PNGase (Png1p) and the 26 S proteasome was found to be mediated via the protein Rad23 (10.Suzuki T. Park H. Kwofie M.A. Lennarz W.J. J. Biol. Chem. 2001; 276: 21601-21607Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), further indicating the functional relationship of the deglycosylating enzyme with the proteasomal degradation. However, up to now knowledge about the structural features of this enzyme has been very limited. PNGase has been classified as a member of the “transglutaminase superfamily” based on the conservation of amino acid residues surrounding the catalytic triad of transglutaminase (11.Makorova K.S. Aravind L. Koonin E.V. Prot. Sci. 1999; 8: 1714-1719Crossref PubMed Scopus (142) Google Scholar). Transglutaminases catalyze amide bond formation between the γ-carboxyamide group of glutamine and the ε-amino group of lysine (12.Lorand L. Conrad S.M. Mol. Cell Biochem. 1984; 58: 9-35Crossref PubMed Scopus (662) Google Scholar). The cross-linking of the two proteins involves deamidation of glutamine. Png1p catalyzes a reaction (cleavage of an amide bond), which is reverse of that in transglutaminases (formation of a amide bond) (Fig. 1). Transglutaminases possess a catalytic triad of three amino acids Cys, His, and Asp. This catalytic triad is similar to that found in thiol proteases (13.Pedersen L.C. Yee V.C. Bishop P.D. Trong I.L. Teller D.C. Stenkamp R.E. Prot. Sci. 1994; 3: 1131-1135Crossref PubMed Scopus (138) Google Scholar). We found that this putative catalytic triad is conserved through all of Png1p homologs, suggesting that Png1p might also utilize this triad for its enzymatic reaction. Based on homology with factor XIIIa and with PNGases from other eukaryotes, we examined several mutations of Png1p at putative active site and non-active site residues. The effect on the Png1p-mediated deglycosylation of a glycopeptide (biochemically purified) supports the idea that Png1p utilizes a similar catalytic triad for an enzymatic reaction, which is consistent with its classification as a novel member of the transglutaminase superfamily. We have shown that Png1p requires Cys-191, which is predicted to supply the nucleophilic residue of the catalytic triad. His-218, which is proposed to function as a catalytic base analogous to the invariant histidine residue present in the catalytic triad of serine and cysteine proteases, is also essential (14.Bazan J.F. Fletterick R.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7872-7876Crossref PubMed Scopus (355) Google Scholar, 15.Gorbalenya A.E. Donchenko A.P. Blinov V.M. Koonin E.V. FEBS Lett. 1989; 243: 103-114Crossref PubMed Scopus (365) Google Scholar). Asp-235, the third residue of the catalytic triad, was also found to be crucial. The carboxylic acid of aspartic acid is important to maintain the proper orientation of the histidine residue, facilitating its participation in catalysis (16.Sprang S. Standing T. Fletterick R.J. Stroud R.M. Finer-Moore J. Xuong N.-H. Hamlin R. Rutter W.J. Craik C.S. Science. 1987; 237: 905-909Crossref PubMed Scopus (202) Google Scholar,17.Craik C.S. Rocznaik S. Largman C. Rutter W.J. Science. 1987; 237: 909-913Crossref PubMed Scopus (286) Google Scholar). Several other residues in Png1p conserved in factor XIIIa have also been identified as essential components for the PNGase activity. The structural and/or functional importance of these mutants in Png1p was examined by comparison with the known three-dimensional structure of factor XIIIa (18.Yee V.C. Pedersen L.C. Trong I.L. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acd. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (323) Google Scholar). The mutagenic oligonucleotide primers were designed based on the amino acid sequence of yeast Png1p. All residues were mutated to Ala (one at a time) except Cys-129, Cys-132, Cys-165, and Cys-168, which were mutated either to Ala or Ser, and Trp-220 and To-231, which were mutated either to Ala or Phe. Mutagenesis was performed by using a site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's procedure. The primers used are summarized in Table I. All mutations were confirmed by DNA sequencing.Table IPrimers used in this study Open table in a new tab DNA manipulations were performed according to Sambrook et al. (19.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Constructs made were transformed into BL21(DE3)pLysS cells and expression of PNG1 and mutants were induced by adding 1 mmisopropyl β-d-thiogalactoside at A600 = 0.8/ml. After 3 h at 37 °C, 3 ml of cells was centrifuged and protein was extracted by adding 400 μl of phosphate-buffered saline, 1% Triton X-100, 1 mmdithiothreitol (DTT), and 1 mm phenylmethylsulfonyl fluoride, followed by sonication on ice using a Branson sonicator at level 3 twice for 10 s with a cooling interval of 1 min. The cell extract was centrifuged at 16,000 × g for 10 min at 4 °C, and the supernatant was assayed for PNGase activity. All the protein extracts were analyzed on 10% SDS-PAGE. The protein amount was also checked by Western blot analysis. Protein extracts were resolved on SDS-PAGE and transferred to nitrocellulose membranes. Blots were incubated with 1:1000 dilution of rabbit anti-(his)6antibody (Santa Cruz Biotechnology, Santa Cruz, CA), followed by 1:1000 dilution with anti-rabbit IgG horse radish peroxidase-conjugated secondary antibody (Roche Molecular Biochemicals, Indianapolis, IN). Gels were visualized using chemiluminescence to PNGase activity was assayed using as T. A. S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, T. Park H. Lennarz W.J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). was on a CA) and using further the and structural features of the and the Png1p and Ser, Ser, Ser, Ser, Trp-220 and were in as The from a was with ml of 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 10 mm β-mercaptoethanol and the cells were using a The extract was subjected to for h the supernatant to a was with according to the manufacturer's and with the mm and 10 mm The protein was to for h at 4 The amount was then transferred to a and the was The was further with the of and and then with 3 of mm to 1 were After and were to each to the to be 1 mm were analyzed by SDS-PAGE and assayed for PNGase activity. The were and was using a 10 with mm 10 mm and 1 mm for circular dichroism protein were using 10 and mm 1 10 mm and 1 mm All the protein were for at The protein was at using an for for and for All were on a A cell was used to data in the and All the were at The of all the was for and for were with a of and were in mm 1 mm and 10 mm The of a was from all Png1p and mutants and were used to the and of the using glycopeptide as a The enzymatic reaction was performed in mm 1 mm the of was to a reaction up to 1 min for and up to h for mutants when mm of was was out at of the and for 1 min for and h for reaction were as of the of The of the present was to the of of the conserved amino acid residues of Png1p based on the sequence homology with factor XIIIa, a member of the transglutaminase three-dimensional structure is known (18.Yee V.C. Pedersen L.C. Trong I.L. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acd. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (323) Google Scholar). The protein sequence of Png1p was with PNGases and factor XIIIa using the (Fig. In the present study Png1p from was the sequence is shown at the for factor XIIIa is shown at the The mutations on Png1p and the activity with are in Table the activity, the proteins were as and proteins were well and analyzed by SDS-PAGE. Protein expression for mutants of putative catalytic residues His-218, and and proteins from cysteine mutants in two Cys-132, Cys-165, and are shown in 3 and shown in 3 and these proteins were also by Western using activity of mutants used in the acid activity to with activity these mutants activity was h of at shown for other mutants were min of as and for is to these mutants activity was h of at shown for other mutants were min of as and for is to these mutants activity was h of at shown for other mutants were min of as and for is to these mutants activity was h of at shown for other mutants were min of as and for is to activity these mutants activity was h of at shown for other mutants were min of as and for is to Open table in a new tab of and catalytic residues by 10% SDS-PAGE. for each was for and by of and two by 10% SDS-PAGE. for each as and was for the and by Western of and using rabbit antibody to protein Western of and using rabbit antibody to protein The Cys-191, His-218, and Asp-235 of Png1p are well conserved residues throughout the Png1p and were with the catalytic triad, and of factor XIIIa (Fig. and were to the effect of each of these residues on enzymatic activity. The of and proteins were similar based on SDS-PAGE (Fig. as well as Western blot using antibody (Fig. deglycosylation of the was with any of the three proteins which is in with enzyme (Fig. and Our data the that the that Png1p is a novel member of the transglutaminase superfamily (11.Makorova K.S. Aravind L. Koonin E.V. Prot. Sci. 1999; 8: 1714-1719Crossref PubMed Scopus (142) Google Scholar), all of which a similar catalytic triad to out enzymatic This is a common type of enzymatic catalysis in cysteine proteases J. PubMed Scopus Google Scholar), serine proteases D. Nature. PubMed Scopus Google Scholar, C.S. J. Nature. PubMed Scopus Google Scholar), and M. S.M. M. I. J. J.L. A. Protein PubMed Scopus Google Scholar). Other the catalytic residue several other acid mutants that enzyme activity were identified by these were mutants residues and were to a protein amount to was used for enzyme activity and mutants were found to be However, of each of the Trp-220 and to residue, retained enzyme activity The Trp-220 and be to and of factor XIIIa, These suggest that these residues are in the structural stability of the This is consistent with the the catalytic triad and residues, other residues, and of which are with and of factor XIIIa, were also found vitally important for the activity of Png1p and are involved in the formation of a bond in factor XIIIa. the loss of activity with mutants or is due to the conformation of the structure of the enzyme as confirmed by analysis. This to that a similar bond might be in Png1p. Png1p are sequence the “transglutaminase (9.Suzuki T. Park H. Hollingsworth N. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1052Crossref PubMed Scopus (190) Google Scholar). these conserved residues are cysteine residues that two Cys-132, Cys-165, and in (9.Suzuki T. Park H. Hollingsworth N. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1052Crossref PubMed Scopus (190) Google Scholar). The mutations of these residues resulted in loss of the enzymatic activity However, to an enzyme with a very level of activity. the other residues and are also conserved in PNGases from are conserved in factor These residues from the catalytic residues were found to be for enzyme activity activity. of also that this is an essential residue in the native structure of enzyme these residues, of the other conserved residues in any effect on enzyme activity further the relationship of the and and and were of protein was used for all the The (Fig. at and and a at of proteins with a and in with the data on the secondary structure of Png1p Koonin E.V. Aravind L. Mol. 2001; 10: PubMed Scopus Google Scholar). However, loss in was with the The of a protein be to the of secondary structure components N. Biochem. PubMed Scopus Google Scholar). of the secondary structure of and Png1p is shown in Table with the have a in the of the was in the of into may have the of the of the of and in and Png1p using N. Biochem. PubMed Scopus Google or Open table in a new tab was also used to study the in the conformation of the and The of protein and which are to and residues, (Fig. However, the when proteins and were used (Fig. These are consistent with a non-native conformation for the Png1p protein, and the loss in enzyme activity is due to disruption of the native structure of the enzyme. The in Table that for mutants and activity was min of However, these mutants revealed very level of activity, other mutants further effect on activity. The of these mutants to and the with and mutants to two at the in all Png1p (9.Suzuki T. Park H. Hollingsworth N. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1052Crossref PubMed Scopus (190) Google Scholar). These mutants were as and The of all protein were analyzed by using 10% SDS-PAGE (Fig. The amount of protein in these to mutants was to analyzed by using Western blot (Fig. the of an amount of protein in the to the activity was in all the mutants with The of all to residues were with enzyme. shown in Table a was found in and of with However, the was similar for the and these Png1p. The activity of mutants in the suggest that the bond of the amino acid cannot the bond of cysteine residue at that of the of to mutants of or for is to × × × × for is to Open table in a new tab PNGase activity has been in the degradation of proteins (7.Wiertz E.J.H.J. Jones T.R. Sun L. Bogyo M. Geuze H.J. Ploegh H.L. Cell. 1996; 84: 769-779Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, 8.Wiertz E.J.H.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Crossref PubMed Scopus (955) Google Scholar, T. Park H. Hollingsworth N. Sternglanz R. Lennarz W.J. J. Cell Biol. 2000; 149: 1039-1052Crossref PubMed Scopus (190) Google Scholar, T. Park H. Kwofie M.A. Lennarz W.J. J. Biol. Chem. 2001; 276: 21601-21607Abstract Full Text Full Text PDF PubMed Scopus (105) Google Rapoport T.A. Ploegh H.L. J. Cell Biol. 1999; PubMed Scopus Google Scholar, T. S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, Ploegh H.L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, H. T. Lennarz W.J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). it has been reported that yeast Png1p with the 26 S proteasome through Rad23 protein, a protein (10.Suzuki T. Park H. Kwofie M.A. Lennarz W.J. J. Biol. Chem. 2001; 276: 21601-21607Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). the of Png1p has been found to proteasome proteins, the idea that Png1p is involved in proteasomal degradation H. T. Lennarz W.J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). Based on structure Png1p has been reported to be a member of the transglutaminase superfamily of proteins (11.Makorova K.S. Aravind L. Koonin E.V. Prot. Sci. 1999; 8: 1714-1719Crossref PubMed Scopus (142) Google Scholar). In the structure-function relationship of Png1p was further investigated by amino acid residues that are essential for enzyme activity. The catalyzed by these in transglutaminase superfamily either the formation of by to the of proteins or of (11.Makorova K.S. Aravind L. Koonin E.V. Prot. Sci. 1999; 8: 1714-1719Crossref PubMed Scopus (142) Google Scholar). Png1p in the transglutaminase superfamily is consistent with the reaction that involves of the amide bond between and an N. Biochem. PubMed Scopus Google Scholar). The three-dimensional structure of Png1p is the structural effects of the mutations were predicted using the three-dimensional structure of factor XIIIa (18.Yee V.C. Pedersen L.C. Trong I.L. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acd. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (323) Google Scholar). In is shown the structural of the catalytic and other conserved residues in factor XIIIa. The catalytic residue in factor XIIIa, which is shown to be essential for the catalytic activity J. Biol. Chem. Full Text PDF PubMed Google Scholar, C.S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), is at the amino of the in the The group of a bond with catalytic C.S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, R. R. W. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), which from a in the of a The of a bond with the other of the is on the of the The bond interaction between and is that of a as in the cysteine The loss in activity of Png1p of any of the three catalytic residues His-218, and to may be due to the of between Cys-191, His-218, and Asp-235 the structure which in effects charge in bond the which is consistent with of the non-native conformation for the enzyme. Our are consistent with the loss in activity for all the three catalytic residues when mutated to at a in factor XIIIa C.S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). the conserved residues in residues Trp-220 and Trp-231, which are also conserved in factor XIIIa (Fig. a loss of enzyme activity on to A for this be based on the known structure of factor XIIIa. The residues in factor XIIIa are and is and to the catalytic residue and has with the of the orientation of this catalytic residue (Fig. conserved residue, is found in the of 3 and with the of the of The importance of these between the catalytic and Trp-220 and the of and in Png1p was by of Trp-220 and to an residue Phe. of these mutants retained enzyme activity. In factor XIIIa, the of when on the its in the formation of a bond with a of to the of in The residues in Png1p are and In factor XIIIa, these residues are to be in to the of and/or to Ala this bond 1 and to a non-native conformation of the This based on the sequence mapping in structure of factor XIIIa, is consistent with the of These for a bond between and in Png1p. The of two and other residues and in Png1p of these residues are found to be conserved in factor XIIIa. is to the and structural importance of the and the other residues. all of that mutations within the of the enzyme have a on the structure of protein, with proper of the enzyme. The present data that the fold of the of Png1p might be similar with the other of the transglutaminase suggesting a common evolutionary lineage Koonin E.V. Aravind L. Mol. 2001; 10: PubMed Scopus Google Scholar). The of this study was the of catalytic amino acid residues Cys-191, His-218, and Asp-235 in which are involved in the cleavage of amide bond between and the in a These catalytic residues are well conserved in all which to the catalytic similar to the mechanism of serine proteases, in the deglycosylation reaction as shown in The of a is by Cys-191, which the group of the of to glycan a this is the the of up the and a This process is then by the effect of the of all the three residues be involved in charge which is essential for the catalytic The to an the is the in the which a the aspartic acid is at the site of cleavage The which is the of is to glycan and as shown in This is the study on the of Png1p. structure or structural are to further the structure-function relationship of and this of study is We of the Lennarz for and for the is a to N. from Cold Spring Harbor Laboratory for