©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Sequence of Porcine Protein NH-terminal Asparagine Amidohydrolase
A NEW COMPONENT OF THE N-END RULE PATHWAY (*)

(Received for publication, October 21, 1994; and in revised form, November 9, 1994)

Albert E. Stewart Stuart M. Arfin Ralph A. Bradshaw (§)

From the Department of Biological Chemistry, School of Medicine, University of California, Irvine, California 92717-1700

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Co- and post-translational amino-terminal processing of proteins is one mechanism by which intracellular proteins can be either protected from or targeted to degradation by the N-end Rule pathway (Bachmair, A., Finley, D., and Varshavsky, A.(1986) Science 234, 179-186). A novel enzyme, protein NH(2)-terminal asparagine amidohydrolase, which can function in this pathway by potentially directing critical regulatory proteins possessing an amino-terminal asparagine residue formed from the removal of N-acetylmethionine, has recently been purified and characterized (Stewart, A. E., Arfin, S. M., and Bradshaw, R. A.(1994) J. Biol. Chem. 269, 23509-23517). Here, we report the isolation and characterization of a cDNA for porcine protein NH(2)-terminal asparagine amidohydrolase, which indicates that it is a new type of enzyme, not homologous to any previously identified protein. This provides strong evidence for the importance of regulated protein degradation in cellular functioning.


INTRODUCTION

Eukaryotic proteins with asparagine (as well as aspartic and glutamic acids) in the penultimate position retain the initiator methionine and become N-acetylated as a cotranslational event (1, 2, 3) (Fig. 1). From data base analyses, some 43 unique (nonspecies-redundant) proteins beginning with the Met-Asn- sequence have been identified(4) . These proteins are almost exclusively involved in such regulatory functions as transcription, signal transduction, and developmental processes. Such proteins are often rapidly turned over, by as yet uncharacterized degradation pathways, in order to allow tight control over the steady-state level of activity. From previous studies(5, 6, 7) , it is known that exposure of the penultimate asparagine (or aspartic or glutamic acid) residue destabilizes proteins, rendering them susceptible to N-end Rule-mediated polyubiquitinylation and degradation by the proteasome. The deblocking step is presumably catalyzed by an acylamino-acid hydrolase that removes the N-acetylmethionine (Fig. 1). However, neither amide nor acid side chains are recognized by the ubiquitin-protein ligase alpha (E3alpha), which has two independent binding sites for basic and large hydrophobic residues, respectively(7, 8, 9) . Such recognition is a prerequisite for the polyubiquitinylation step. In the case of proteins with either aspartic or glutamic acid at the NH(2) terminus, recognition is achieved by the addition of an NH(2)-terminal arginine via arginyl-tRNA:protein transferase (10, 11) . Proteins with NH(2)-terminal asparagine are not substrates for arginyl-tRNA:protein transferase, and the asparagine must be converted to aspartic acid for them to be handled by this pathway. We developed an assay to detect such activity and have identified and purified to homogeneity an enzyme, protein NH(2)-terminal asparagine amidohydrolase (PNAA), (^1)from porcine liver extracts that specifically deamidates NH(2)-terminal asparagine residues(4) . (^2)The enzyme does not act on substrates with internal or COOH-terminal asparagines and does not act on glutamine residues in any position. In this report, we describe the cDNA and predicted protein sequence and report the interesting observation that, like its activity, it represents a unique protein, unrelated to any known sequence at a detectable level.


Figure 1: Proposed pathway for the co- and post-translational modification of eukaryotic proteins commencing with the sequence Met-Asn-. NAT, N-acetyltransferase; AAH, acylamino-acid hydrolase; PNAA, protein NH(2)-terminal asparagine amidohydrolase; PRT, arginyl-tRNA:protein transferase; E2, ubiquitin-conjugating enzyme; E3alpha, ubiquitin-protein ligase alpha; Ubq, ubiquitin; M, methionine; N, asparagine; R, arginine; D, aspartic acid. The dashedline represents the messenger RNA coding for the indicated sequence; the solidlines represent protein. The brackets indicate the hypothetical role proposed for PNAA.




MATERIALS AND METHODS

CNBr Digestion and Peptide Fractionation

PNAA was prepared as described previously(4) . The enzyme (3.3 µg) was dissolved in 70% (v/v) trifluoroacetic acid (50 µl), and an excess of CNBr was added. The mixture was purged with argon and placed in the dark for 20 h. The reaction mixture was loaded directly onto a C(4) column (2.1 times 30 mm) attached to a Model 140 solvent delivery system (Applied Biosystems, Inc., Foster City, CA) equilibrated in buffer A (0.1% trifluoroacetic acid in H(2)O) at a flow rate of 0.2 ml/min. Peptides were eluted with a linear gradient to 100% buffer B (0.07% trifluoroacetic acid in acetonitrile) over 60 min. Absorbance at 214 nm was monitored.

Protein and Peptide Sequencing

Native PNAA and PNAA-derived CNBr peptides were sequenced on a Model 477A pulsed-liquid sequenator with an on-line phenylthiohydantoin analyzer (Applied Biosystems, Inc.). Peak heights were quantitated by the software of the system.

Molecular Biology Methods

All molecular biology protocols were performed according to standard procedures (12, 13) unless otherwise noted.

Oligonucleotides

Synthetic oligonucleotides were prepared by solid-phase methods on an Applied Biosystems Model 391A DNA synthesizer using cyanoethyl phosphoramidite-derivatized nucleotides.

PCR Primers

5`-RACE primers were synthesized according to the method of Frohmann et al.(15) : gene-specific primer 1 (GSP-1) antisense, 5`-GCTCTGTAGATCCTCCGCAGTCTTCACGTTGAC-3; and GSP-2 nested antisense, 5`-AGTGGTTTTCATTTTCTTCCCG-3`. gt10 EcoRI site-flanking PCR primers were as follows: forward, 5`-CAAGTTCAGCCTGGTTAAGTC-3`; and reverse, 5`-TATGAGTATTTCTTCCAGGGT-3`.

Preparation of Poly(A)-enriched Porcine Liver RNA

Total RNA was prepared by the method of Chomczynski and Sacchi (14) from 1 g of freshly excised porcine liver (Farmer John, Los Angeles, CA). Poly(A)-enriched RNA was prepared by oligo(dT)-cellulose (Boehringer Mannheim) chromatography.

First Strand cDNA Synthesis from Porcine Liver mRNA

First strand cDNA synthesis using Superscript II reverse transcriptase (Life Technologies, Inc.) from poly(A)-enriched porcine liver RNA (5 µg) was primed with oligo(dT) by the method of Frohmann et al.(15) .

PCR with Degenerate Oligonucleotide Primers

First strand cDNA (100 ng) was used as template in a 50-µl reaction mixture with 20 pmol of appropriate primer pairs (see Fig. 2), 67 mM Tris (pH 8.8 at room temperature), 6.7 mM MgCl(2), 16.6 mM (NH(4))(2)SO(4), 10 mM beta-mercaptoethanol, 10% (v/v) Me(2)SO, and a 1.25 mM concentration of each deoxynucleoside triphosphate. After one step each of denaturation (95 °C, 6 min) and annealing (cooling to 25 °C over 20 min, followed by an increase to 45 °C over 10 min), 2 units of Taq polymerase were added, and the temperature was held at 45 °C for 2 min and then at 72 °C for 8 min. Samples were then incubated through 40 cycles of 94 °C for 1.75 min and 45 and 72 °C for 2 min at each temperature, with a final extension at 72 °C for 4 min. Amplified products were subcloned into the PCR-II vector (Invitrogen, San Diego, CA) following the manufacturer's instructions.


Figure 2: Design of degenerate PCR oligonucleotides based on PNAA amino acid sequences. CNBr-1 and CNBr-2 are cyanogen bromide cleavage-derived peptides of PNAA. -Fold degeneracy is given in parentheses. Left-to-right arrows and right-to-left arrows indicate the sense (5`-3`) and antisense (3`-5`) orientations, respectively. I is inosine. X indicates a position in which full codon degeneracy is represented.



DNA Sequencing and Sequence Analysis

Plasmid DNA sequencing was by the chain termination method using a modified T7 DNA polymerase and [alpha-S]dATP with the Sequenase 2.0 kit (U. S. Biochemical Corp.). DNA sequence analysis was performed with the Wisconsin Genetics Computer Group software package (16) on line at the University of California Irvine Convex cluster and by electronic mail server through the BLAST software package provided by the National Center for Biotechnology Information(17) .

Northern Blot Analysis

The 615-bp PCR fragment was labeled using the Multiprime DNA labeling system (Amersham Corp.) with [alpha-P]dCTP. Poly(A) mRNAs (5 µg each) from rat pheochromocytoma PC12 cells and rat brain (kindly provided by Dr. Simona Raffioni) and porcine liver were electrophoresed on a 1% formamide/formaldehyde-agarose gel and blotted onto GeneScreen nylon membrane (DuPont NEN) by standard methods. Prehybridization was for 12 h at 42 °C in 750 mM NaCl, 5 mM EDTA, 50 mM NaHPO(4) (pH 7.4), 50% formamide, 5 times Denhardt's solution, 10% dextran sulfate, 1% SDS, and 100 µg/ml salmon sperm DNA. Hybridization (5 times 10^6 cpm PCR probe) was for 22 h at 42 °C. The membrane was washed with 2 times SSPE (standard sodium phosphate with EDTA) for 20 min at room temperature, followed by three 20-min washes at 55 °C in 0.1 times SSPE, 2% SDS. The blot was exposed to Kodak XAR-5 film for autoradiography.

gt10 cDNA Library Screening

Phage and Escherichia coli host strain C600-hfl were propagated and plated on NZYDT medium (Life Technologies, Inc.). A porcine liver gt10 cDNA library (CLONTECH) was plated at a density of 35,000 plaque-forming units/150-mm plate. Phage plaques were lifted onto nitrocellulose filters (Schleicher & Schuell) by standard procedures. Filters were prehybridized for 12 h at 42 °C in 50% formamide, 5 times SSC, 50 mM NaHPO(4) (pH 6.8), 0.1% sodium pyrophosphate, 2 times Denhardt's solution, and 10 µg/ml salmon sperm DNA. Hybridization (30 times 10^6 cpm PCR probe) was carried out for 22 h at 42 °C. Filters were washed twice at room temperature in 2 times SSC, 0.1% SDS for 20 min, followed by three washes at 42 °C in 0.2 times SSC, 0.1% SDS. Duplicate positive clones were identified by autoradiography, plaque-purified phage DNA was prepared by standard methods, and insert sizes were determined by PCR analysis using primers flanking the EcoRI cloning site.

5`-RACE

5`-RACE was performed as described by Frohmann et al.(15) . First strand cDNA synthesis from poly(A) RNA (5 µg) using Superscript II reverse transcriptase was primed with GSP-1 (20 pmol) at 48 °C. 5`-RACE PCR reactions were performed with 5 µl of 5`-RACE reaction, GSP-2 nested primer (20 pmol), AP-1 primer (20 pmol), and (dT) adapter primer (10 pmol). PCR reaction conditions were 1.5 min each of denaturation (95 °C), annealing (57 °C), and extension (72 °C) for 24 cycles. RACE products were ligated to the PCR-II vector. Four clones of each product were selected at random and sequenced completely on both strands.


RESULTS AND DISCUSSION

Partial amino acid sequence data were obtained by direct pulsed-liquid sequenator analysis of 50 pmol of high performance liquid chromatography-desalted porcine PNAA and of four CNBr fragments ( Fig. 2and Fig. 3). Clearly, CNBr-1 is derived from the NH(2) terminus of the protein, indicating that proline is the penultimate residue that is exposed by the cotranslational processing of the nascent protein by methionine aminopeptidase(2) . However, CNBr-2 also had an NH(2)-terminal proline that could have arisen during the CNBr cleavage from acid-catalyzed hydrolysis of an Asp-Pro bond(18) . This was shown not to be the case by sequence analysis of PNAA treated with only 70% trifluoroacetic acid, which produced only the single sequence corresponding to CNBr-1. The fractions containing CNBr-3 and CNBr-4 were actually mixtures of CNBr-1 and CNBr-2, respectively. Their sequences could, however, be accurately deduced by subtraction. Nonetheless, CNBr-1 and CNBr-2 were selected for preparing oligonucleotide primers because the sequence data for these peptides were considered to be more reliable.


Figure 3: Full-length porcine liver PNAA cDNA clone. The full-length sequence of PNAA constructed by combining the gt10 library-derived cDNA and the 5`-region with the initiator methionine derived from 5`-RACE is shown. Underlined amino acid sequences correspond to those derived from sequencing of CNBr-1-4 peptides. The arrows indicate the positions and orientations of the PCR primers used to amplify the 615-bp fragment from porcine liver first strand cDNA as discussed in the text. The box-arrow shows the 5`-end of the isolated gt10 cDNA clone. Boldface italics indicate the 5`- and 3`-stop codons, initiator methionine codon, and polyadenylation signal.



Eight degenerate oligonucleotide primers were prepared corresponding to both orientations from each end of CNBr-1 and CNBr-2 (Fig. 2). PCR was used to amplify a cDNA fragment from porcine liver poly(A) mRNA. An 600-bp fragment was obtained using the PN-2/PN-5 primer pair. As expected, no products were obtained using the PN-1/PN-6 or PN-3/PN-8 primer pair, which would only produce a fragment if the order of CNBr-1 and CNBr-2 were reversed in the protein. Further positive controls indicated the authenticity of the 600-bp fragment. When the PN-2/PN-5 reaction product was used as template for the primer pairs PN-2/PN-7 and PN-5/PN-8, 564- and 54-bp fragments were produced, respectively, as predicted by amino acid sequence analysis. The PN-2/PN-5 fragment contained a 615-bp open reading frame with all of the CNBr fragment sequences (Fig. 3) and resolved a few amino acid sequencing ambiguities of CNBr-1 (data not shown).

Utilizing the 615-bp sequence as a probe, three clones from a recombinant phage gt10 porcine liver cDNA library were identified and sequenced. One clone (400 bp) had a 285-bp open reading frame (also present in the 615-bp PCR fragment) flanked on the 5`- and 3`-ends by untranslated sequence. In the absence of consensus intron-exon boundaries, it was assumed to be a library artifact. The remaining two clones of 756 bp were shown to be identical and encompassed 567 bp of open reading frame (encoding 189 amino acids) and 189 bp of 3`-untranslated region including a polyadenylation signal (AATAAA) 19 bp upstream from a 29-bp poly(A) tail.

Although the continuous reading frame produced by the overlapping PCR and cDNA clones likely represented the full protein sequence (because the 5`-end of the PCR fragment began with the sequence corresponding to the NH(2) terminus of the protein), 5`-RACE was employed to establish the 5`-untranslated sequence upstream from the putative initiation site. First strand cDNA prepared by reverse transcription of poly(A) RNA primed with a 5`-gene-specific oligonucleotide (based on the cDNA clone) produced products in the expected size range of 400-500 bp (based on a 1-kilobase RNA transcript determined by Northern analysis; see below) with gene-specific and nested RACE primers. Two cDNA RACE products of 460 and 672 bp were isolated and sequenced. These exactly overlapped the cDNA clone (and the PCR fragment) and contained 33 bp of 5`-untranslated sequence with a putative initiator methionine in the expected position. The upstream sequence (CGCGAGATGCCG) reflects a rarely used start site as judged by Kozak's rules(19) . The presence of two in-frame stop codons immediately upstream from the designated translational start site lends support to this assignment. Subsequent analyses with higher stringency reverse transcription demonstrated that the shorter fragment (460 bp) arose from reverse transcriptase ``jumping.''

As shown in Fig. 3, a full-length sequence for porcine PNAA can be assembled from these data. In addition to the 5`-untranslated sequence, there is an open reading frame of 930 bp encoding 310 amino acids. The deduced composition is in good agreement with that determined for the whole protein(4) , including the 4 methionine residues that formed the basis of the CNBr strategy, and the calculated molecular mass of 34,760 Da is in excellent accord with the values of 33,000 and 34,000 Da observed by reducing SDS-polyacrylamide gel electrophoresis and gel filtration, respectively(4) .

Northern analysis, using the 615-bp PCR fragment as a probe, was carried out with poly(A)-enriched RNAs from pig liver, rat brain, and rat PC12 cells. As shown in Fig. 4, a well resolved transcript of 1 kilobase was identified. The levels of transcript appear to be much higher in the two neuronal sources (PC12 cells can be induced to differentiate into cells with a phenotype and morphology closely resembling those of sympathetic neurons(20) ). However, the cellular origins of these transcripts are not yet known, and thus, the significance of these observations is not presently clear. Whether they suggest lower levels of potential N-end Rule-mediated turnover (5) in liver as opposed to brain remains to be determined.


Figure 4: Northern analysis of PC12 cell, rat brain, and pig liver poly(A)-enriched RNAs with a 615-bp PNAA PCR fragment. mRNAs (5 µg each) from PC12 cells, rat brain, and pig liver (lanes A-C, respectively) were electrophoresed on a 1% formamide/formaldehyde-agarose gel and blotted onto GeneScreen nylon membrane. The subcloned 615-bp PNAA PCR product (5 times 10^6 cpm) was used as a probe. The blot was hybridized for 22 h at 42 °C in 750 mM NaCl, 5 mM EDTA, 50 mM NaHPO(4) (pH 7.4), 50% formamide, 5 times Denhardt's solution, 10% dextran sulfate, 1% SDS, and 100 µg/ml salmon sperm DNA. The blot was washed at 55 °C in 0.1 times SSPE. Ribosomal RNAs (28 and 18 S) are indicated by arrows.



A comparison of both the nucleotide and predicted amino acid sequences of porcine PNAA with the nonredundant GenBank data base and the Expressed Sequence Tag data base revealed no significant similarity to any entry. We particularly examined E. coli asparaginases I (21) and II (22) for more detailed comparison because these enzymes perform similar chemical transformations (albeit asparaginase acts only on free asparagine and PNAA only on NH(2)-terminal asparagines in peptide linkage). These enzymes have similar molecular masses (35,388 and 34,080 Da, respectively) as PNAA (34,760 Da), although both prokaryotic enzymes are tetrameric and PNAA is monomeric(4, 21, 22) . No significant relatedness was observed. However, similarity may still be found at the three-dimensional level when the structures of these (or other) enzymes are determined.

Although the strategy to isolate and characterize PNAA(4) , ultimately leading to the cloning and sequence determination described herein, was based on a presumed role in the N-acetylmethionine-linked pathway of the N-end Rule (23) (Fig. 1), we have no direct evidence that PNAA participates in this pathway or that its activity is limited to this role. It is possible that it may direct the activation (or inactivation) of peptides or proteins that remain to be identified. Such pathways do not necessarily depend on acylamino-acid hydrolase, as NH(2)-terminal asparagines could be generated by proteolysis of precursor structures. For the most part, these are thought to be extracellular events, and PNAA appears to be an intracellular protein, but such processes may still be found to occur inside cells. There is significant intracellular proteolytic capacity, such as manifested in the calpains, the physiological and cellular relevance of which remains largely unknown(24) . However, not withstanding such possibilities, the most likely role for PNAA remains its participation in the regulated turnover of N-acetyl-Met-Asn proteins. Given the nature of the proteins that bear this structure(4) , this may eventually prove to be among the most important functions of the N-end Rule.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant DK-32465 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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)/EMBL Data Bank with accession number(s) U17062[GenBank].

§
To whom correspondence should be addressed. Tel: 714-824-6236; Fax: 714-824-8036; rablab{at}uci.edu.

(^1)
The abbreviations used are: PNAA, protein NH(2)-terminal asparagine amidohydrolase; RACE, rapid amplification of cDNA ends; GSP, gene-specific primer; PCR, polymerase chain reaction; bp, base pair(s).

(^2)
This enzyme was originally designated protein NH(2)-terminal asparagine deamidase(4) , but has been herein called by the more systematically correct amidohydrolase.


ACKNOWLEDGEMENTS

We are grateful to M. Boguski (National Center for Biotechnology Information) for assistance in screening the nucleic acid/protein sequence data base. We thank S. Raffioni, S. Disper, M. Kobrin, and S. Grigoryev for helpful discussions and A. Xie for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.