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(Received for publication, October 21, 1994; and in revised form, November 9, 1994) From the
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 Eukaryotic proteins with asparagine (as well as aspartic and
glutamic acids) in the penultimate position retain the initiator
methionine and become N
Figure 1:
Proposed pathway for the co- and
post-translational modification of eukaryotic proteins commencing with
the sequence Met-Asn-. NAT, N
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.
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
Figure 3:
Full-length porcine liver PNAA cDNA clone.
The full-length sequence of PNAA constructed by combining the
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
Utilizing the 615-bp
sequence as a probe, three clones from a recombinant phage 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 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
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
A comparison of both the
nucleotide and predicted amino acid sequences of porcine PNAA with the
nonredundant GenBank 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 The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U17062[GenBank].
Volume 270,
Number 1,
Issue of January 6, 1995 pp. 25-28
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-terminal Asparagine Amidohydrolase
A NEW COMPONENT OF THE N-END RULE PATHWAY (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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-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.
-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
(E3),
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 terminus, recognition is achieved by the addition of an
NH-terminal arginine via arginyl-tRNA:protein transferase (10, 11) . Proteins with NH-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-terminal asparagine amidohydrolase (PNAA), (
)from porcine liver extracts that specifically deamidates
NH-terminal asparagine residues(4) . ()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.
-acetyltransferase; AAH,
acylamino-acid hydrolase; PNAA, protein
NH
-terminal asparagine amidohydrolase; PRT,
arginyl-tRNA:protein transferase; E2, ubiquitin-conjugating
enzyme; E3, ubiquitin-protein ligase ; 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.
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
column (2.1 
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 HO)
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, 16.6 mM (NH)SO, 10 mM 
-mercaptoethanol, 10% (v/v) MeSO, 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.
DNA Sequencing and Sequence Analysis
Plasmid DNA
sequencing was by the chain termination method using a modified T7 DNA
polymerase and [-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
[-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
(pH 7.4), 50% formamide, 5
Denhardt's solution, 10% dextran sulfate, 1% SDS, and 100
µg/ml salmon sperm DNA. Hybridization (5 10
cpm
PCR probe) was for 22 h at 42 °C. The membrane was washed with 2
SSPE (standard sodium phosphate with EDTA) for 20 min at room
temperature, followed by three 20-min washes at 55 °C in 0.1
SSPE, 2% SDS. The blot was exposed to Kodak XAR-5 film for
autoradiography.
Phage and Escherichia coli host strain C600-hfl were propagated and
plated on NZYDT medium (Life Technologies, Inc.). A porcine liver
gt10 cDNA Library Screening
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
SSC, 50 mM NaHPO (pH 6.8),
0.1% sodium pyrophosphate, 2 Denhardt's solution, and 10
µg/ml salmon sperm DNA. Hybridization (30 10 cpm PCR probe) was carried out for 22 h at 42 °C. Filters
were washed twice at room temperature in 2 SSC, 0.1% SDS for 20
min, followed by three washes at 42 °C in 0.2 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.
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-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.
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.
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).
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.
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.''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.
10 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 (pH 7.4), 50% formamide, 5
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
SSPE. Ribosomal RNAs (28 and 18 S) are indicated by arrows.
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
-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.-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.
)-terminal asparagine amidohydrolase; RACE, rapid
amplification of cDNA ends; GSP, gene-specific primer; PCR, polymerase
chain reaction; bp, base pair(s).)-terminal asparagine
deamidase(4) , but has been herein called by the more
systematically correct amidohydrolase.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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