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Blood, 15 August 2000, Vol. 96, No. 4, pp. 1596-1598
BRIEF REPORT
Human erythrocyte pyrimidine 5'-nucleotidase, PN-I, is
identical to p36, a protein associated to lupus inclusion formation in
response to  -interferon
Adolfo Amici,
Monica Emanuelli,
Nadia Raffaelli,
Silverio Ruggieri,
Franca Saccucci, and
Giulio Magni
From the Istituto di Biochimica and Dipartimento di
Biotecnologie Agrarie ed Ambientali, Università di Ancona,
Ancona, Italy.
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Abstract |
Erythrocyte maturation is accompanied by RNA degradation and
release of mononucleotides. We have previously purified PN-I, a
pyrimidine nucleotidase whose deficiency is associated with hemolytic
anemia. Computer-aided analysis of PN-I tryptic and CNBr peptide
sequences revealed substantial identity with tryptic peptide sequences
reported for p36, an  -interferon-induced protein. PN-I partial
sequences were matched through the expressed sequence tag database with
different human complementary DNA (cDNA) clones, whose sequences were
exploited to screen a human placenta cDNA library. PN-I cDNA, coding
for a 286-residue protein, was expressed in Escherichia
coli, yielding a fully active recombinant enzyme. The recombinant
protein sequence comprised the peptide sequences determined for PN-I
and p36. Rabbit antisera raised against two peptides deriving from p36
and PN-I tryptic digestions, respectively, recognized both wild-type
and recombinant PN-I. Molecular properties of the two proteins were
essentially the same. We conclude that p36 and PN-I are identical proteins.
(Blood. 2000;96:1596-1598)
© 2000 by The American Society of Hematology.
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Introduction |
Erythrocyte pyrimidine 5'-nucleotidases are enzymes
involved in the salvage pathway of nucleotides and in the catabolism of RNA, in that they catalyze the dephosphorylation of various pyrimidine nucleoside monophosphates to their respective nucleosides. Two human
red cell soluble enzymes, termed PN-I and PN-II, were identified on the
basis of their different molecular properties and substrate specificities.1-7 The deficiency of PN-I is associated to
a hemolytic nonspherocytic anemia characterized by basophilic stippling
at the Wright stain and accumulation of pyrimidine
nucleotides,1 suggesting that in the red cell the
fluctuation of pyrimidine levels is differently regulated with respect
to its purine counterpart. Evidence has been reported on the
accumulation of ribonucleic material as the cause of the basophilic
stippling, indicating that PN-I deficiency is also closely related to
incomplete RNA degradation. Recently it has been described that rat
PN-I-like enzyme activity increased concomitantly with the maturation
of new erythrocytes,8 suggesting a major role of this
enzyme in the elimination, as diffusible nucleosides, of the pyrimidine nucleotides formed from internal RNA degradation. Likewise, in red
blood cells of human fetuses from 17 to 23 weeks of gestation, the
activity of pyrimidine 5'-nucleotidase was higher than in the adult
cells.9 A transient increase of a similar activity has
also been observed in chick embryonic erythrocytes in response to
cyclic adenosine monophosphate stimulation of the enzyme
synthesis.9 Furthermore, PN-I deficiency has been
associated to the conversion of hemoglobin E disease into an unstable
hemoglobinopathy-like disease.10
We have previously demonstrated that homogeneous pyrimidine
nucleotidases from human erythrocytes possess phosphotransferase activities specific for pyrimidine nucleotides,11
suggesting an additional role of these enzymes in nucleotide
metabolism. To get insights into the physiological importance of PN-I,
we decided to investigate on the molecular properties of the enzyme. In
the present study the enzymatic protein was submitted to chemical and
enzymatic digestions, and it has been shown that the sequences of the
resulting peptides, when searched in protein data bank, matched with
those of six peptides deriving from digestions of p36, an
-interferon-induced protein in cells forming lupus inclusions (LIs).12 LIs are abnormal cytoplasmic structures of
unknown function, constituted by membranes, proteins, carbohydrates,
and RNA.13 The PN-I complementary DNA (cDNA) was for the
first time cloned and expressed. Its inspection reveals a sequence
comprising, in addition to those stored in data bank, other sequences
separately and independently determined both for p3612 and
PN-I. Furthermore, rabbit antisera raised against synthetic peptides
constructed on p36 and PN-I sequences cross-reacted with PN-I. These
results indicate that p36 and PN-I are identical proteins.
| |
Study design |
A total of 10 µg homogeneous PN-I11 was both CNBr-
and trypsin-digested.14 The fragments were separated on a
Capillary Microblotter system and sequenced on an Applied Biosystems
Procise sequencer.
Human placenta library was used as the source of PN-I cDNA, which was
amplified for cloning by using polymerase chain reaction (PCR). The
product, digested with BamH I and Pst I, was
ligated into the expression vector pT7-7. Escherichia coli
BL21, harboring the recombinant plasmid, was grown at 37°C, in
luria-bertani (LB) medium. At different incubation times, cells
were collected and disrupted by sonication. Samples were assayed for
PN-I activity.11
Rabbit antisera raised against the peptides KSSVRIKNPTRVEEC
and DNSNIILLGDSQGDC were obtained from Igtech (Salerno, Italy), according to standard procedure. The immunoreactive protein was detected as previously described.15
| |
Results and discussion |
Partial sequences obtained from PN-I digestions were used for
similarity searches, using the NCBI BLAST server. In the "nr" database, the PN-I sequences matched with the 82-residue sequence obtained for p36,12 with the exception of only three
residues (Figure 1a). In this regard, it
should be pointed out that two of the residues are at the p36 peptide
terminal positions, whose identification generally is more prone to
ambiguity, and the third (Q) is just the deamidated form of the other
(E). A search of the GenBank expressed sequence tag database (dbEST),
using program "tblastn," revealed a perfect match of all
PN-I-derived peptides with several anonymous cDNA sequences. Each
single cDNA sequence coded only for a part of the protein, and it was
needed to overlap and combine the sequences to obtain the entire
putative nucleotide cDNA sequence coding for PN-I, as well as the
untranslated regions. Moreover, the translation of the combined cDNA
into amino acid sequence made it possible to align the tryptic and CNBr
peptide sequences, totalling 251 amino acids of the coded sequence. It should be pointed out that the PN-I-deduced amino acid sequence (Figure
1b) also comprises p36 T42 (LQIITDFDM) and T50 (YM) residues, which are
not present in the sequence determined for PN-I in our laboratory. This
finding represents an additional evidence of the identity of p36 and
PN-I proteins, and it is reinforced by the observation that the pI
value of 5.4 calculated for the recombinant PN-I is superimposable to
that of 5.6 reported for p36.12
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| Figure 1.
Comparison of PN-I and p36 sequences.
(A) Alignment of p36 sequences, as reported,12 with those
determined for PN-I. (B) Human PN-I cDNA sequence and its encoded amino
acid sequence. The first base of the translation start codon is
designated +1 and the stop codon is indicated by asterisks. The 251 amino acids obtained from PN-I peptide sequencing are underlined.
Residues of fragments T42 and T50 of p36 that are not aligned with PN-I
peptides (panel A) are boxed in the PN-I sequence.
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To clone and express PN-I cDNA, amplification experiments were carried
out using a human placenta library. On the basis of the PN-I sequence,
primers delimitating the cDNA portion of interest were chosen and added
to specific restriction sites for cloning as described in the section
"Study design." PCR amplification yielded a product of 880 base
pairs (bp), whose nucleotide sequence exactly corresponded to the data
bank sequence. The expression resulted in a soluble and active PN-I.
Such activity was not detectable in control extracts prepared from BL21
cells transformed with the nonrecombinant plasmid (not shown). In the
cells harboring the recombinant plasmid, a spontaneous and transient
expression of PN-I activity at the late logarithmic phase was found,
followed by a rapid decrease at the stationary phase (Figure
2a). Sodium dodecyl sulfate
polyacrylamide gel electrophoresis of the same extracts revealed the
appearance of a 36 000-Mr protein band, whose intensity
paralleled the activity expression behavior (not shown).
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| Figure 2.
Expression of PN-I activity in
E coli and PN-I immunoblot analysis. (A) The
specific activity of the expressed PN-I ( ) is plotted as a function
of growth of BL21 cells transformed with the recombinant plasmid  .
(B) Antiserum raised against peptide KSSVRIKNPTRVEEC is incubated at
1:1500 dilution with blotted pure wild-type PN-I (lane 1); antiserum
against peptide DNSNIILLGDSQGDC is incubated at 1:1500 dilution with
blotted pure wild-type PN-I (lane 2), and blotted extracts of BL21
cells transformed with nonrecombinant (lane 3) and recombinant plasmid
(lane 4).
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To further ascertain whether PN-I and p36 indeed represent the same
protein, rabbit antibodies were raised against two synthetic peptides
constructed on the basis of two sequences: KSSVRIKNPTRVEEC, localized
at the PN-I N-terminus, and DNSNIILLGDSQGDC, identical to the sequence
of the p36 T29 fragment. The antibodies were able to recognize both
wild-type and recombinant PN-I, when analyzed by Western blot (Figure
2b), thus confirming both the expression of the recombinant protein and
its identity with p36.
The significance of the identity of the two proteins is at the moment
obscure. Computer-aided search of the 286-amino acid sequence revealed
high similarity with hypothetical proteins of Caenorhabditis
elegans and Arabidopsis thaliana,
suggesting a conserved function of biological importance. We do not
know whether p36 retains the ability to catalyze the hydrolysis of
pyrimidine monophosphates. The occurrence of posttranslational
modifications of p36, not affecting the apparently identical molecular
and structural parameters but influencing the catalytic properties,
cannot be discounted. However, our results might open a new possible
scenario on the role played by p36 in immune diseases, in that LI form in the cytoplasm of reticuloendothelial cells of individuals with systemic lupus erythematosus 16 and acquired
immunodeficiency syndrome.17 The availability of cDNA,
together with full structural and catalytic characterization of the
protein, now actively in progress in our laboratory, will be
instrumental for more systematic experiments aiming at the elucidation
of the function of PN-I in normal and pathological conditions.
| |
Footnotes |
Submitted January 3, 2000; accepted April 13, 2000.
Supported in part by CNR grant 98.00473.ct04 and CNR Target Project
"Biotechnology."
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Giulio Magni, Istituto di Biochimica,
Facoltà di Medicina e Chirurgia, Università di Ancona,
Via Ranieri 65, 60131 Ancona, Italy; e-mail: magnig{at}popcsi.unian.it.
| |
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Abstract
Introduction