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Articles |
1
School of Allied Health Sciences, Tokyo Medical and Dental University, 5-45, Yushima 1-chome, Bunkyo-ku, Tokyo 113-8519, Japan.
2
Department of Human Life Science, Jissen Women’s
University, 4-1-1, Ohsakaue, Hino City, Tokyo 191-8510, Japan.
a Author for correspondence. Fax 81-3-5803-0161; e-mail
m.kawai.mtec{at}med.tmd.ac.jp.
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Abstract |
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Methods: Reverse transcription-PCR and direct sequencing were performed with leukocyte CD13 mRNA from the propositus. Two-dimensional electrophoresis and N-terminal amino acid sequencing were performed with serum CD13 from the propositus, the father of the propositus, and healthy volunteers.
Results: The sequence of the CD13 cDNA of the propositus was essentially identical with that reported previously. However, the CD13 polypeptide of the propositus and the father of the propositus was truncated, lacking amino acids 1–43 of intact CD13 (43-truncated CD13), whereas CD13 lacking residues 1–58 (58-truncated CD13) and 43-truncated CD13 were detected in serum from healthy volunteers.
Conclusions: In serum from healthy volunteers, we found both 58-truncated CD13, a major isoform reported previously, and 43-truncated CD13, a novel, minor isoform with a larger polypeptide. In serum of the family, 43-truncated CD13 was extremely concentrated, suggesting that proteolytic cleavage of CD13 amino acids 43 and 44 (43-truncation) is abnormally promoted. Because no mutation was found in the CD13 cDNA from the propositus, increased serum CD13 in this family seems to be caused by a mutation in a gene that regulates 43-truncation protease activity.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The catalytic domain of CD13 faces the exterior of the plasma membrane and is anchored by a transmembrane-spanning domain, which is composed of N-terminal residues 9–32 (2). However, an N-terminal truncated form of CD13 lacking residues 1–58 (58-truncated CD13) has been purified from 300 mL of normal serum (3), suggesting that some proteolytic cleavage mechanism may release a small amount of truncated CD13 into serum of normal individuals. Because the molecular mass of CD13 polypeptide of the propositus should be larger than that of healthy individuals, it also seems possible that an abnormality in proteolysis of CD13 may exist, cleaving the anchored CD13 polypeptide at a position closer to the NH2 terminus to produce a longer, unanchored polypeptide. In this study, we investigated whether the larger CD13 polypeptide of this family with high serum aminopeptidase N (CD13) activity is attributable to a mutation in the CD13 gene or to an abnormality in posttranslational modification of the CD13 polypeptide.
Aminopeptidase N is the enzyme identical to CD13 based on the cDNA sequence (4). In this report, we use "CD13" to refer to the "CD13/aminopeptidase N" protein and "aminopeptidase N" to refer to the enzyme activity of CD13.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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preparation of leukocyte mRNA FROM THE PROPOSITUS
Whole blood (20 mL) was collected from the propositus in a tube
containing heparin and mixed with 13 mL of a solution containing 20 g/L
dextran and 0.154 mol/L NaCl; the tube was then incubated at room
temperature for 20 min. The upper layer of plasma was transferred to a
new tube. Ficoll-Conray solution (5 mL) was gently layered under the
plasma, and the tube centrifuged at 1000g for 30 min. The
cell pellet was suspended in 20 mL of 0.033 mol/L NaCl, mixed with 20
mL of 0.27 mol/L NaCl, and centrifuged at 1000g for 5 min.
Total RNA was extracted from the cell pellet according to the acid
guanidinium thiocyanate-phenol-chloroform method described by
Chomczynski and Sacchi (5). The first extraction step was
initiated by adding 0.8 mL of D solution, 80 µL of 2 mol/L sodium
acetate (pH 4.0), 0.8 mL of phenol, and 160 µL of chloroform to the
pellet. This extraction yielded 3.2 µg of RNA from the leukocytes of
the propositus.
reverse transcription-pcr and direct sequencing
Complementary DNA synthesis was performed with the SuperScript
Preamplification System (Life Technologies) according to the
manufacturer’s procedure for first-strand cDNA synthesis. The reaction
mixture contained 1.0 µg of RNA from the propositus and
oligo(dT)12–18 in a total volume of 10 µL.
Reverse transcription-PCR was performed in the regions numbered
1–8 with the respective forward and reverse primers as shown in Table 1
. The regions overlap partially and together span the entire
translated region of CD13 mRNA (nucleotides 121–3024). The PCR
reaction mixture (10 µL) contained 0.5 µL of the
reverse-transcription reaction and a final concentration of the
following components: 10 mM Tris-HCl (pH 8.3), 1.5 mM
MgCl2, 50 mM KCl, 0.2 mM deoxyribonucleoside
triphosphates (dATP, dCTP, dTTP, and dGTP), 1.0 µM forward and
reverse primers (shown in Table 1
), and 0.1 U/µL AmpliTaq DNA
Polymerase (PE Biosystems). The amplification was performed in a
FTS-4000 Capillary Thermal Cycler (Corbett Research) using the
following cycling program: 30 cycles of 94 °C for 20 s, each
annealing temperature (shown in Table 1
) for 20 s, and 74 °C
for 40 s, with a final extension at 72 °C for 5 min. The PCR
product was electrophoresed on a 5% polyacrylamide gel and
stained with 0.5 mg/L ethidium bromide. The bands considered for
analysis were excised from the gel, ground, and shaken vigorously in
water. After centrifugation, the supernatant was subjected to a second
round of PCR amplification in a volume of 100 µL and electrophoresed.
PCR product was electroeluted from the excised gel and precipitated
with ethanol.
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Sequencing of the PCR products was performed with a 370A DNA sequencer (PE Biosystems) and a Dye Terminator Cycle Sequencing Ready Reaction reagent set (PE Biosystems) according to the supplier’s instructions. Oligonucleotides 1105–1125 and 1533–1554 were also used as forward primers for sequencing the reverse transcription-PCR products. Nucleotide numbers used here are based on the report by Look et al. (4).
CD13 from the serum of healthy volunteers was partially purified by DEAE-cellulose ion-exchange column chromatography and ammonium sulfate precipitation. DEAE-cellulose column chromatography was performed as described previously (1). Eluted sample was dialyzed against 10 mmol/L sodium phosphate (pH 7.2) overnight. Ammonium sulfate was then added to each sample at a final concentration of 2.3 mol/L. The mixture was stirred for 30 min and centrifuged at 20 000g for 30 min. To the supernatant, ammonium sulfate was added to achieve a final concentration of 3.1 mol/L; the mixture was then stirred and centrifuged again. The precipitate was dissolved in 10 mmol/L sodium phosphate (pH 7.2) and dialyzed overnight against 10 mmol/L sodium phosphate (pH 7.2), 0.137 mol/L NaCl. Ammonium sulfate precipitation and dialysis were performed at 4 °C.
From 6 mL of serum, 
500 µL of partially purified sample was
obtained. The specific activity of aminopeptidase N in serum vs the
partially purified sample in a representative experiment was 0.81 U/g
of protein vs 4.7 U/g of protein. Approximately 50% of the
aminopeptidase N activity was recovered. Aminopeptidase N activity was
assayed according to the procedure described previously (1),
and 1 U represents the activity that hydrolyzes 1 µmol of
L-leucine-p-nitroanilide per minute.
The protein concentration was measured with BCA protein assay reagents
(Pierce).
immunoprecipitation of serum cd13
Immunoprecipitation was carried out on serum from an affected
member of the family with high serum aminopeptidase N (CD13) activity
or partially purified CD13 from a healthy volunteer. CD13 was
immunoprecipitated from 500 µL of sample with 75 µL of mouse
monoclonal human CD13 antibody MCS-2 (100 mg/L; Nichirei) and 5 µL of
rabbit anti-mouse IgG 
-globulin (28.7 g/L) according to the
procedure described previously (1).
ief, sds-page, and n-terminal amino acid sequence analysis
For combined IEF and SDS-PAGE (two-dimensional electrophoresis),
an immunoprecipitated sample was dissolved in 20 µL of 8 mol/L urea,
20 g/L Ampholine (preblended, pH 3.5–9.5; Amersham Pharmacia Biotech),
and 20 g/L Triton X-100. With a MiniPROTEAN Tube Cell apparatus
(Bio-Rad Laboratories), first-dimensional IEF was performed on a 4%
polyacrylamide capillary gel containing the above components; the gel
was subjected to 500 V for 10 min followed by 1500 V for 3 h. The
electrophoresed capillary gel was equilibrated with sample buffer and
subjected to a second dimension of SDS-PAGE, using a 3% polyacrylamide
stacking gel (1 cm length, 1 mm thickness) and a 5% separating gel
(6-cm length), according to the procedure described by Laemmli
(6). The proteins were transferred to ProBlott
polyvinylidene difluoride (PVDF) membranes (PE Biosystems) at 2
mA/cm2 for 1 h (7) and then
visualized by staining with Coomassie Brilliant Blue R-250.
One-dimensional SDS-PAGE and blotting were also performed with an
immunoprecipitated sample, which was dissolved in 20 µL of sample
buffer containing 0.1 mol/L dithiothreitol.
The spot or band with a molecular mass of 144 kDa was excised and analyzed with an HPG1005A amino acid sequencer (Hewlett Packard) or a Procise 494 cLC (PE Biosystems). The amino acid sequences, as determined, were compared with those in the SwissProt, SwissProt-upd, PIR, PRF, and PDBSTR databases, which were accessed through GenomeNet (http://www.fasta.genome.ad.jp/) with the FASTA program.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Relative to the previous report by Look et al. (4), an A-to-G base change was detected at nucleotide 1929, which is within the translated region and produces an amino acid change at residue 603 from Ile to Met. The variability in amino acid 603 was reported in the sequence of aminopeptidase N (8), which was identified as CD13 (4). Thus, a SNP (homozygote) at nucleotide 1929 produces a neutral substitution at amino acid 603 that is not likely to affect the function of the molecule. A G-to-C base change was also detected at nucleotide 3053 in the 3'-untranslated region. These results indicate that the predicted amino acid sequence of CD13 of the propositus is very similar to the sequence from the previous report (4).
n-terminal amino acid sequencing of serum cd13 from the propositus
and father of the propositus
Serum from the propositus was subjected to immunoprecipitation and
then analyzed with two-dimensional electrophoresis (Fig. 1
, left panel). As reported previously (1), the
protein with a molecular mass of 144 kDa was recognized as CD13.
Because this protein was broadly extended in the direction of IEF,
three membrane pieces (A, B, and C) were excised from this band (Fig. 1
, right panel). Amino acid sequencing of these three samples revealed
identical N-terminal sequences (Ser-Pro-Val-Ala-Ser) that exactly
coincided with the predicted residues 44–48 of intact CD13 (Fig. 2
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Immunoprecipitated CD13 from serum of the propositus’ father was
separated by one-dimensional SDS-PAGE (Fig. 3
). Amino acid sequencing of the excised band revealed the
N-terminal sequence (Ser-Pro-Val-Ala-Ser-X-Thr/Ser-Pro-Ser-Ala, where X
is an unidentified residue), which coincided with the residues 44–53
of CD13 (Fig. 2
). A search of the SwissProt, SwissProt-upd, PIR, PRF,
and PDBSTR on-line databases (accessed in August 2000) for
human proteins with our peptide sequence (the father of the propositus)
yielded the strongest match with CD13 amino acids 44–53. Thus, the
identity of the band recognized as CD13 was confirmed from its amino
acid sequence. These results clearly show that serum CD13 from the
affected members of this family is almost entirely composed of a
truncated form of CD13 lacking residues 1–43. This novel CD13 isoform
was designated 43-truncated CD13.
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n-terminal amino acid sequencing of serum cd13 from healthy
volunteers
CD13 was purified from the serum of each healthy volunteer as
described in Materials and Methods. The sample from
volunteer 1 was analyzed with two-dimensional electrophoresis and
stained with Coomassie Brilliant Blue (Fig. 4
, left panel). The band recognized as CD13 seemed to consist of
two spots that partially overlapped. Two pieces of PVDF membrane, piece
A, with a lower pI, and piece B, with a higher pI, were excised (Fig. 4
, right panel) and sequenced. Identical analyses were performed on
samples from volunteers 2 and 3. In addition, 36 mL of serum was
collected from volunteer 3 for longer sequencing.
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The results of the N-terminal amino acid sequencing are shown in Table 2
. Relative to the polypeptide predicted from the cDNA nucleotide
sequence, CD13 amino acid residues 44–48 were detected in serum from
volunteer 1, residues 44–47 were detected in serum from volunteer 2,
and residues 44–51 were detected in serum from volunteer 3 (Table 2
and Fig. 2
). Unexpectedly, these results indicate that normal serum
also contains 43-truncated CD13, although at a relatively low
concentration.
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In addition, CD13 amino acid residues 59–63 were detected in volunteer
1 and residues 59–66 were detected in volunteer 3 (Table 2
and Fig. 2
). These results indicate that normal serum contains another truncated
isoform of CD13 lacking N-terminal amino acids 1–58 (58-truncated
CD13), as reported previously (3). The N-terminal amino acid
sequencing results for samples from volunteer 2 were consistent with
this interpretation. A search of the databases mentioned earlier
(accessed in August 2000) for amino acid sequences with the
same sequence as CD13 amino acids 59–66 (volunteer 3) was negative,
indicating that the protein excised from PVDF membranes after IEF and
SDS-PAGE was authentic CD13.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Watanabe et al. (3) purified 58-truncated CD13 from 300 mL of serum, a combined sample that was collected from nonpregnant healthy individuals. In the present study, we confirmed the presence of 58-truncated CD13 in each serum sample from two nonpregnant, healthy individuals. Moreover, we demonstrated the presence of 43-truncated CD13, a novel isoform, in normal serum, although at a low concentration. Because 58-truncated CD13 was the only truncated isoform purified from normal serum in the study by Watanabe et al. (3), it seems likely that 58-truncated CD13 is the major isoform and 43-truncated CD13 is a minor isoform in normal serum. In contrast, 43-truncated CD13 was the only isoform detected in serum from the propositus and the father of the propositus. Although 58-truncated CD13 may also exist as a minor component in these individuals, there seems to be no doubt that 43-truncated CD13 is the major component. Thus, extremely high concentrations of the 43-truncated isoform are the cause of the high aminopeptidase N (CD13) activity in the serum of the propositus and her father.
In our previous investigation, SDS-PAGE of serum CD13 treated with neuraminidase, N-glycosidase, and O-glycosidase indicated that the CD13 polypeptide of the propositus should be larger than that of an unrelated healthy individual (1). Because the cDNA sequences of individuals with normal and high serum aminopeptidase N (CD13) activity are nearly identical, the size difference of the CD13 polypeptide between healthy and affected individuals is likely to be attributable to N-terminal cleavage at different locations in the peptide sequence. This hypothesis was substantiated by the results showing that the serum CD13 of the propositus and her father is composed predominantly of 43-truncated CD13, an isoform with a longer polypeptide, whereas 58-truncated CD13 is probably a major isoform in the serum of healthy individuals.
As reported previously (1), serum aminopeptidase N (CD13) activity was extremely high in the propositus, the father, and the paternal grandmother of the propositus of the family (affected members), whereas the serum activity was normal in the mother and the sister of the propositus (normal members; autosomal dominant). IEF under nondenaturing conditions revealed that a pI 3.3 isoform was the predominant component of serum CD13 in the affected members, whereas a pI 4.1 isoform was the dominant serum isoform in the normal members and an unrelated healthy individual. Thus, we termed the CD13 isoform with pI 3.3 as a variant CD13; the extremely high concentrations of this variant CD13 are the cause of the high serum aminopeptidase N (CD13) activity of the family. The results of N-terminal amino acid sequencing indicate that variant CD13 (pI 3.3) must be 43-truncated CD13, whereas the pI 4.1 isoform is likely to be 58-truncated CD13. Consistent with this assumption, the isoelectric point of 43-truncated CD13 was somewhat lower than that of 58-truncated CD13 under denaturing conditions (8 mol/L urea and 20 g/L Triton X-100). In terms of cDNA sequence and N-terminal truncation, the variant CD13 itself does not deserve to be called "variant" because 43-truncated CD13 also exists in normal serum. However, an extraordinarily high concentration of 43-truncated CD13 in serum is a specific trait of the affected members of the family.
Our previous IEF analysis of neuraminidase-treated CD13 indicated that the sialic acid content of the variant CD13 is higher than normal (1). The extra peptide portion consisting of amino acid residues 44–58 in 43-truncated CD13 does not contain negatively charged amino acid residues or possible N-glycosylation sites for sialylated carbohydrates (4). However, CD13 carries O-glycans predominantly located in the Ser/Thr-rich residues 43–65 (9). Therefore, a few Ser or Thr residues in the extra portion of 43-truncated CD13 may be O-glycosylated with sialylated carbohydrates, which should produce the lower pI (3.3) of 43-truncated CD13.
Because the CD13 molecule is cleaved at specific positions adjacent to the transmembrane domain, truncated forms of serum CD13 probably result from a proteolytic cleavage mechanism. A model for this hypothesis is the transferrin receptor, in which the NH2 terminus also serves as a transmembrane anchor. Like CD13, the serum form of the transferrin receptor is truncated N-terminally and lacks this anchor (10). A cell membrane-associated serine protease is likely to cause truncation of the transferrin receptor (11). Alternatively, the truncated CD13 polypeptides may be true secretory forms of CD13 that are products of either multiple genes or alternative mRNA splicing pathways, like the secretory forms of HLA antigens (12) and IgM (13). However, no reports have supported this alternative hypothesis (4)(8)(14)(15)(16). Therefore, the formation of these truncated isoforms of CD13 is likely to be a result of proteolytic cleavage by protease.
Watanabe et al. (17) suggested that CD13 truncation may occur in a tissue-specific manner because sera from pregnant women contained an isoform lacking residues 1–69 (69-truncated CD13), which is likely to be derived from the placenta, and the 58-truncated CD13 was found in most nonpregnant individuals (3). We showed that sera from nonpregnant healthy individuals contain at least two isoforms, 43- and 58-truncated CD13. This fact indicates that different kinds of CD13 proteolysis (43- and 58-truncation of CD13) occur in healthy individuals. Moreover, it is likely that different proteases (43- and 58-truncation protease), which may be tissue-specific, catalyze CD13 proteolysis.
CD13 is a cell surface marker of granulocytes, monocytes, and their progenitors. CD13, also known as aminopeptidase N, exists in other tissues, including kidney, liver, and small intestine (2). Secretion of CD13 from cells can be caused by loss of its N-terminal transmembrane domain (18). For example, Madin-Darby canine kidney cells transfected with cDNA corresponding to CD13 lacking amino acid residues 1–64 secreted this form of CD13 into the medium (18). However, the final destination of intracellularly transported CD13 is the apical (luminal) membrane in polarized cells. After synthesis, direct sorting of CD13 to the apical membrane occurs in kidney cells (18)(19)(20). To be released into serum, CD13 should appear on the basolateral membrane (sinusoidal side) before apical transport. In hepatocytes, this indirect sorting of CD13 via the basolateral membrane actually occurs (21), and in enterocytes, CD13 seems to be transported partially via the basolateral membrane (22)(23). In these cells, 43- or 58-truncation of CD13 during its transport to the basolateral membrane should lead to the release of CD13 into serum. Thus, truncation of CD13 should have an essential role in releasing CD13 into serum from hematopoietic cells, hepatocytes, and possibly enterocytes, which seem to be the major sources of serum CD13 in healthy individuals and in members of the family studied here with high serum aminopeptidase N (CD13) activity.
CD13 generally is accepted as an integral membrane protein in which the signal peptide (residues 9–32) is not removed but is retained as its transmembrane anchor (5)(24). In nonpregnant, healthy individuals, proteolysis of CD13 must be maintained at a relatively low level. Therefore, the release of CD13 from the surface of cells is a minor pathway, and consequently, serum aminopeptidase N (CD13) activity remains low. However, in the family of the propositus, serum aminopeptidase N activity is much higher than that of healthy individuals and mainly consists of the 43-truncated CD13. Therefore, proteolysis of CD13 between amino acid residues 43 and 44 (43-truncation) seems to be increased in some tissue. As reported previously, a mutation of a particular gene, which is inherited in autosomal dominant mode, is responsible for high serum aminopeptidase N (CD13) activity in this family (1). It seems likely that a gene that regulates 43-truncation protease activity is mutated, leading to increased 43-truncation of CD13.
CD13 has been suggested to play an important role in the pathophysiology of several diseases. For example, CD13 on various human metastatic tumor cell lines mediates the migration of those tumor cells through Matrigel (25)(26). CD13 is also expressed to degrade kinins and neuropeptides on the surface of synovial cells derived from rheumatoid or osteoarthritic joints (27). Concerning regulation of CD13, several factors that up-regulate CD13 expression have been reported, including DMP1 (28). However, the mechanism responsible for down-regulation of CD13 on the cell surface remains poorly understood. The protease catalyzing 43- or 58-truncation of CD13 may play an important role in several pathophysiological mechanisms through down-regulation of CD13 on the cell surface.
The reason that the affected members of the family discussed here do not have any particular symptom or disease remains unknown. One possibility is that the source organ cells that release large amounts of 43-truncated CD13 into the serum of the affected members may produce enough CD13 to compensate for the increased release of CD13 from the cells.
In conclusion, we identified a novel isoform of CD13 that is N-terminally truncated, lacking amino acids 1–43. At least two different CD13 isoforms, 43-truncated CD13 and 58-truncated CD13, were detected in serum from healthy volunteers. This observation indicates that at least two different truncating proteases of CD13 exist and are likely to be expressed in different tissues. In the family with high serum aminopeptidase N (CD13) activity, there was no meaningful mutation in the CD13 gene itself. However, we showed that high serum aminopeptidase N (CD13) activity is predominantly derived from the 43-truncated isoform, suggesting increased 43-truncation in this family. Therefore, a gene regulating 43-truncation of CD13 may be mutated.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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