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Prepublished online as a Blood First Edition Paper on June 28, 2002; DOI 10.1182/blood-2002-01-0045.
IMMUNOBIOLOGY
From the Department of Internal Medicine and Molecular
Science, Graduate School of Medicine, Osaka University, and Department
of Pathology, Osaka University Medical School, Osaka,
Japan; Pharmaceutical Development Laboratory and
Pharmaceutical Research Laboratory, Kirin Brewery Co Ltd, Takasaki,
Gunma, Japan; and Oklahoma Medical Research Foundation,
825 Northeast, 13th St, Oklahoma City.
Several reports have described "multifunctional"
eukaryotic mRNAs producing more than one protein through alternative
translational initiation at multiple AUG codons. There are 2 such
codons in the 5' region of our recently cloned limitin gene
where 2 open reading frames overlap by 34 nucleotides. The deduced
protein translated from the first ATG contains 33 amino acids, lacks a signal peptide, and has no obvious effects on the transfected 293T
cells. We found that the second ATG is more effective as a
translational initiation site than the first ATG and yields a secreted
protein of 182 amino acids with the same activity as products made with
full-length limitin cDNA. Immunohistochemical and reverse
transcription-polymerase chain reaction analysis revealed that the
longer limitin protein is produced by mature T lymphocytes in spleen
and thymus as well as by bronchial epithelial and salivary duct cells
in healthy mice. Properties of recombinant limitin were determined,
revealing it to be a serologically distinct, heat- and acid-stable,
heparin-binding glycoprotein with the potential for dimerization.
Although the longer limitin protein is structurally and
characteristically related to type I interferons, its production is
uniquely regulated by translation as well as transcription.
(Blood. 2003;101:178-185) B-lymphocyte precursors undergo immunoglobulin (Ig)
heavy- and light-chain gene rearrangements within bone marrow, and only cells with useful receptors are selected for export to the periphery. The extensive proliferation and differentiation events that accompany this vital process are controlled by signals from the
microenvironment.1-6 For example, the orientation and
movement of maturing lymphocytes is dependent on transmembrane cell
interaction molecules displayed by marrow stromal
cells.1,7,8 Culture studies have revealed that stem cell
factor, Flt3 ligand, and interleukin 7 (IL-7) are sufficient positive
signals for survival and differentiation of murine lymphocyte
precursors.9-11 Results of experimental animal studies
indicate that steroid hormones are important for limiting the numbers
of lymphocytes that are produced under normal and steady-state
conditions.12-14 A recently discovered cytokine that we
designated limitin is also a potential negative regulator of B
lymphopoiesis.15 The deduced limitin sequence has homology with interferon It is interesting that the 5' portion of the limitin cDNA
contains 2 ATGs, with 2 corresponding open reading frames that overlap by 34 nucleotides (Figure 1). The first
could encode a 33-amino acid peptide that would lack a signal sequence.
It could be speculated to have intracellular roles and translation is
now demonstrated in this study. Translational initiation at the second
of the 2 ATGs would result in a completely different 182-residue
secreted protein. Experiments were performed to learn the efficiency of such translation and to authenticate the protein product as
biologically active limitin. Although the AUG codon nearest the 5' end
of mRNA is usually a unique site for initiation of translation and most mRNAs in eukaryotes are monocistronic, some mRNAs for proto-oncogenes, transcription factors, and growth factors are regulated at the translational level. Some transcripts can encode 2 or more proteins via
alternative translational initiation at different AUG
codons.16-23 The functional relevance of such complex
arrangements is not well understood, but usually entails some
regulation or inhibition. The potent biologic activity of limitin
requires that it be produced under rigid and tissue-specific control.
We now provide information about the relative translation efficiency
from its 2 potential initiation sites. To better understand limitin, we
also examined its source in healthy mice and its protein character
using newly established polyclonal antibodies specific to the
longer limitin protein that is translated from the second ATG.
Cells and mice
Balb/c mice were purchased from Nippon SLC (Shizuoka, Japan). All mice
were maintained at the Institute for Experimental Animals, Osaka
University. To purify CD4+, CD8+, and
CD45RA/B220+ cell populations, peritoneal lymph node cells
were positive-selected by immunomagnetic beads conjugated with the
indicated antibodies (Miltenyi Biotec, Auburn, CA), as previously
described.24 In our series, more than 95% of the purified
cells always expressed the indicated phenotypes and their viability was
higher than 98%.
Plasmid construct
To produce proteins that are translated from the first or the second ATG of the limitin gene, limitin cDNA was amplified by PCR. The oligonucleotide primers used for these reactions were as follows: 5'-GGGCTCGAGTCAGCGAGCAAGGAGCCCGAAG-3' and 5'-GGGCTCGAGCTGGGCTGCAGCTCAGCA-3' for the first ATG; 5'-GGGCTCGAGAATCGTCAAGCTTCAGCA-3' and 5'-GGGCTCGAGCTTCTCCTCATCTTGGGC-3' for the second ATG. The amplified fragment was digested with XhoI and cloned into the pEFBOSX plasmid that was yielded by site-directed mutagenesis to remove the XhoI site at 3524 of pEFBOS (1st-ATG/pEFBOSX and 2nd-ATG/pEFBOSX). An Ig/pEFBOSX vector was used to produce Ig fusion proteins composed of CH2 + CH3 cassette of human IgG1.25 The cDNA corresponding to the entire coding region of the limitin protein translated from the second ATG was amplified by PCR with 5'-GGGGCGGCCGCCGCAATCGTCAAGCTTCA-3' and 5'-GGGCTCGAGCTTGGGCCTCTTCTCGCAGA-3', and the PCR sample was digested with NotI and XhoI, and ligated into the Ig/pEFBOSX vector (limitin-Ig/pEFBOSX). Limitin-Ig/pEFBOSX produces a fusion protein composed of the protein translated from the second ATG of the limitin gene and human Ig (limitin-Ig). An HT/pEFBOSX vector was used to produce fusion proteins composed of an HPC4 epitope and a transmembrane domain of tissue factor.25 The cDNA corresponding to the entire coding region of the limitin protein translated from the second ATG was amplified by PCR with 5' GGGGCGGCCGCCGCAATCGTCAAGCTTCA-3' and 5'-GGGCTCGAGCTTGGGCCTCTTCTCGCAGA-3', and the PCR sample was digested with NotI and XhoI, and ligated into the HT/pEFBOSX vector (limitin-HT/pEFBOSX). Preparation of polyclonal antibody specific to limitin (antilimitin) The limitin cDNA except for its signal sequence was amplified by PCR with 5'-CGCAATCGTCAAGCTTCAGCATG-3' and 5'-GAGATTCCTGCACGGGGCTTCTCCTCA-3'. The amplified fragment was digested with NdeI and BamHI and cloned into the pET3a expression vector (limitin/pET3a). Escherichia coli BL21 (DE3) was transformed with the limitin/pET3a plasmid and cultured in the presence of isopropyl thiogalactose (IPTG). The synthesized longer limitin protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the E coli inclusion body and extracted from the gel. Antilimitin antiserum was raised in rabbits that were immunized repeatedly with the purified longer limitin protein.Preparation and purification of recombinant limitin protein translated from the second ATG A pTrace vector (Invitrogen, Groningen, The Netherlands) was used to produce recombinant limitin. The Chinese hamster ovary (CHO) ras no. 1 cells overexpressing recombinant limitin were cultured in DMEM/F-12 medium (Gibco).26 The supernatant was collected after 4 days of culture. The selected recombinant limitin was purified by a combination of immunoaffinity chromatography with antilimitin and ion-exchange chromatography on SP-Sepharose FF (Amersham Pharmacia, Buckinghamshire, England).Detection of amino acid sequence 293T cells were transfected with limitin-Ig/pEFBOSX. Limitin-Ig was purified from the supernatant of the transfectant with a protein A column. Its N-terminal amino acid sequence was determined using a protein sequencer, model 477A (Applied Biosystems, Foster City, CA).Luciferase assay Luciferase assays were performed using the Dual-Luciferase Reporter System (Promega), in which transfection efficiency was monitored by cotransfected pGL-Control vector (Promega), an expression vector of firefly luciferase. The cultured cells were transfected with 10 µg 1st-ATG/pRL-SV40 or 2nd-ATG/pRL-SV40 together with 1 µg pGL-Control vector by lipofectamin transfection method. The transfected cells were lysed in buffer supplied by the manufacturer, followed by measurement of the firefly and renilla luciferase activities on luminometer LB96P (Berthold Japan, Tokyo, Japan). The relative renilla luciferase activities were calculated by normalizing transfection efficiencies according to the firefly luciferase activities.CFU-IL-7 colony assay Bone marrow cells were prepared and suspended in 1 mL assay medium as previously described.27 The semisolid agar assay for colony-forming unit (CFU)-IL-7 was done with 1 ng/mL IL-7. Numbers of colonies were counted after 6 days of culture.Western blot analysis Supernatants or recombinant limitin were subjected to SDS-PAGE, and the proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Immobilion; Millipore, Bedford, MA). After blocking of residual binding sites on the filter, immunoblotting was performed with the antilimitin. Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (Dupont NEN, Boston, MA).RT-PCR Total RNAs (2.5 µg) were reverse transcripted to cDNA at 37°C for 1 hour in total reaction volume of 50 µL comprised of Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT; Gibco), oligo dT (1 µg), 0.1 M dithiothreitol (DTT), 10 µM each dNTP, and 1 × RT buffer. To perform PCR, 10 µL of the above RT mixture was added to PCR buffer containing 1.5 µM MgCl2, 1 U Taq polymerase (Perkin Elmer, Branchburg, NJ), 2 µM each dNTP, and relevant sense and antisense primers. PCR reaction mixtures were amplified under the following conditions: 25 cycles of 94°C for 1 minute, 55°C for 2 minutes, and 72°C for 3 minutes. The oligonucleotide primers used for these reactions were as follows: 5'-TCCAGCGTCCAGCGCAGC 3' and 5'-CGGAGCTCTGCTAGGAAG-3' for limitin, and 5'-CCTAAGGCCAACCGTGAAAAG -3' and 5'-TCTTCATGGTGCTAGGAGCCA -3' for -actin.
Immunohistochemical staining Immunohistochemistry was carried out using paraffin sections of healthy mouse tissues. Deparaffinized sections were incubated in 0.3% hydrogen peroxide in methanol to inactivate endogenous peroxidase. The slides were incubated with antilimitin or anti-CD3 (Dako, Glostrup, Denmark). Binding of the antibody was demonstrated by ENVISION+ system (Dako). As a chromogen, 3, 3'-diaminobenzidine tetrahydrochloride was used.
Preferential translation from the second ATG yields active limitin The 5' portion of the originally isolated cDNA clone for limitin consists of a 997-base pair (bp) insert with ATGs at positions 96 and 161, corresponding to 2 distinct open reading frames (Figure 1). This would presumably allow production of 2 completely different proteins containing 33 or 182 amino acids and only the larger has a predicted signal sequence. To analyze which ATG is the major translation initiation site, we constructed 1st-ATG/pRL-SV40 and 2nd-ATG/pRL-SV40 plasmids that produce renilla luciferase under control of the SV40 early enhancer/promoter (Figure 2A). The renilla luciferase protein is translated from the first ATG of the limitin gene in 1st-ATG/pRL-SV40. Although the 2nd-ATG/pRL-SV40 construct contains both ATGs of the limitin gene, only the second is in-frame for the renilla luciferase protein. The null ATG/pRL-SV40 plasmid whose original ATG for the renilla luciferase protein is destroyed was used for a negative control. As shown in Figure 2B, BMS2.4 cells transfected with 2nd-ATG/pRL-SV40 synthesized renilla luciferase protein more effectively than those transfected with 1st-ATG/pRL-SV40 (> 10-fold). Similar results were observed in all stromal cells examined (BMS2, OP42, and MS-5; data not shown).
The originally isolated clone, full cDNA/pEFBOS, contained full-length
limitin cDNA in the pEFBOS expression vector. We next constructed
1st-ATG/pEFBOSX that produces the shortest limitin gene product and
2nd-ATG/pEFBOSX that produces the larger protein, respectively (Figure
3A). Full cDNA/pEFBOS, 1st-ATG/pEFBOSX,
or 2nd-ATG/pEFBOSX was transfected to 293T cells, and the supernatants of each transfectant were added to cultures of BC7.12 pre-B cells. As
shown in Figure 3, panels B and C, the supernatant of 293T cells
transfected with full cDNA/pEFBOS inhibited cell proliferation and
induced cell death in BC7.12 cells. Whereas the supernatant of 293T
cells transfected with 2nd-ATG/pEFBOSX showed similar growth inhibitory
effects on BC7.12 cells, the supernatant of 293T cells transfected with
1st-ATG/pEFBOSX contained no activity. It is noteworthy that no changes
in morphology or growth were noticed in 293T cells transfected with any
of the constructs. Therefore, no role was apparent for the shortest
limitin gene product and previously described activities can be
attributed to the longer, secreted protein.
Serologic detection and characterization of limitin A polyclonal antibody was prepared by immunizing rabbits with the full-length recombinant limitin protein translated from the second ATG. It recognized 293T cells expressing a fusion protein composed of the longer limitin protein, an HPC4 epitope, and the transmembrane domain of tissue factor, whereas these did not stain 293T cells transfected with a control plasmid (Figure 4A). Western blot analysis with this new reagent revealed that limitin is serologically unique and contains at least one epitope not shared with IFN- , IFN-, or IFN- (Figure
4B). The 293T cells transfected with full cDNA/pEFBOS as well as those
transfected with 2nd-ATG/pEFBOSX expressed full-length limitin protein
that was recognized by antilimitin (Figure 4C). In contrast, no protein
was serologically detected in 293T cells transfected with
1st-ATG/pEFBOSX. Limitin was originally cloned from the BMS2.4 stromal
cell subclone, and this was the only one of 5 cell lines that secreted
an approximately 20-kDa protein (Figure 4D). Supernates of 3 other
stromal cell clones that did not kill pre-B cells and a myelomonocytic
leukemia line that did not express limitin transcripts were negative in
this Western blot assay. These observations establish the specificity of antilimitin, show that the cytokine is serologically distinct from
other IFNs, and confirm that BMS2.4 cells secrete the longer limitin
protein translated from the second ATG.
Limitin expression in normal murine tissues Previous PCR analysis suggested that limitin might normally be transcribed in hematopoietic organs, but little information was available about cell types that produce this new cytokine.15 Thus, we used the new limitin-specific antibody for immunohistochemical staining (Figure 5). Limitin-containing cells were detected in sections of spleen, thymus, lung, and salivary gland. The T cell-rich zone of the spleen known as the periaortic lymphoid sheath included limitin-positive cells. Furthermore, the antibody recognized cells in the thymic medulla where mature CD3+ cells reside. In addition to T lymphocytes, bronchial epithelial cells and salivary duct cells were strongly positive. No limitin protein was detectable in liver, kidney, or heart. With one exception, these results are compatible with PCR analyses made with whole tissue extracts (Figures 5 and 6). Transcripts were detectable in spleen, thymus, lymph node, lung, kidney, and salivary gland, but not in liver or heart (Figure 6A). Note that although limitin mRNA was present in the kidney, no staining was observed by immunohistochemical staining.
A more detailed analysis of limitin expression was then performed with enriched populations of lymphocytes from various tissues. As shown in Figure 6B, RT-PCR detected limitin transcripts in CD4+ as well as CD8+ lymph node cells, but not in samples prepared from CD45RA/B220+ cells. Fetal thymocyte suspensions provided an enriched population of immature double-negative cells, whereas mature CD4+ and CD8+ T lymphocytes emerged gradually after birth (data not shown). As shown in Figure 6C, limitin expression was not detectable in fetal thymocytes (gestation day 14), was very faint in newborn thymuses, and increased in intensity with development. These findings suggest that mature T lymphocytes and epithelial cells, but not B lymphocytes, are a natural source of this cytokine in healthy mice. Structural properties of the secreted limitin protein The ability to express recombinant limitin will facilitate many types of structure-function analysis. The biologic activity of this material was confirmed using CFU-IL-7 colony assays (Figure 7A). Clonal proliferation of B-cell precursors was inhibited in a dose-dependent manner, with maximal activity observed at a concentration of 50 ng/mL. Although IFN- lost
activity when treated with either acid or heat, recombinant limitin was
stable to these treatments (Figure 7B).
The cDNA sequence corresponding to the longer limitin protein predicts a highly hydrophobic stretch at the N-terminal end and biologic activity was found in the supernatant of transfected 293T cells. Although these facts are consistent with a functional signal peptide, we performed N-terminal amino acid sequencing to determine the precise structure of the mature protein. This revealed the sequence LDXGKSGSLH where X represents one undetermined amino acid residue. This precisely corresponds to the Leu22-His31 segment predicted from the limitin cDNA sequence (Figure 1). Therefore, a signal peptide with cleavage site between Ser21 and Leu22 probably facilitates limitin secretion. The limitin sequence contains a single potential N-linked glycosylation
site NXS/T at amino acid residue 68 (Figure 1). As shown in Figure
8A, treatment of recombinant limitin with
N-glycosidase F changed its mobility in SDS-PAGE from 20 to 16 kDa.
Therefore, the longer limitin protein is glycosylated. The limitin
sequence contains 6 cysteine residues, and the mature secreted protein has 5 cysteine residues (Figure 1). Migration was slightly faster in
SDS-PAGE when recombinant limitin was electrophoresed under reducing
rather than nonreducing conditions (Figure 8B). In addition, an
approximately 40-kDa band that was seen under the nonreduced condition
disappeared following 2-mercaptethanol treatment. Therefore, mature
secreted limitin has intramolecular disulfide bonds and may use one
cysteine residue for dimerization.
Chemokines and certain other cytokines physically interact with heparan sulfate proteoglycans of the extracellular matrix. We determined that recombinant limitin bound to heparin-Sepharose (Figure 8C). This recognition was completely blocked by an excess of free heparin, but not chondroitin sulfate. Furthermore, native limitin protein produced by BMS2.4 cells was enriched and purified with a heparin-Sepharose column (data not shown). These observations show that fully active limitin can be produced in recombinant form and extend our understanding of the secreted protein.
This study extends our understanding of the normal expression and biophysical properties of a recently discovered cytokine. The limitin gene contains 2 open reading frames that potentially encode different proteins. However, translational initiation was found to preferentially use the second ATG that encodes the full-length, biologically active cytokine. The protein is serologically distinct from other IFNs and has other characteristics that may be important for its biologic activities. Finally, we show that this potent inhibitor of B lymphopoiesis is constitutively made in healthy animals. Database searches with the limitin cDNA sequence yield few homologous
matches, but many IFNs are retrieved when the analysis is based on the
predicted limitin protein sequence. Structural similarity is also
paralleled by binding and apparent use of the IFN- With the exception of one pseudogene, we are unaware of transcripts for any IFN genes that have 2 ATGs within the 5' segment, as is the case for limitin (Figure 1). Our experiments demonstrate more than 10-fold preference for translational initiation from the second ATG and this results in the secreted, biologically active protein. Although not common, alternative translation sites are used in other eukaryotic genes such as c-myc, cot, glutathione peroxidase, BAG-1/RAP46, catechol O-methyltransferase, HER-2/neu, Pit-1, and molybdopterin synthase.16-23 Multiple proteins translated by alternative initiation from different AUGs were previously shown to yield molecules with discrete functions.16,17 Depending on the cellular environment, expression of 4 isoforms of human BAG-1/RAP46 proteins occurs by alternative translation from 4 different initiation codons.19 Thus, it is important to analyze whether the short limitin gene product has intrinsic activity or only exists to regulate expression of the longer limitin protein translated from the second ATG. Because no obvious changes in morphology or growth were noticed in 293T cells transfected with 1st-ATG/pEFBOSX, it remains unclear if the shortest limitin gene product has a function. Regulation of translational initiation on eukaryotic ribosomes is known
to be complicated, but there is some basis for speculation about how a
particular AUG codon is selected when more than one are
present.32-36 The 40S ribosomal subunit binds initially at the 5' end of mRNA and subsequently migrates to the first AUG codon in
a favorable context for initiating translation. The 5'-proximal AUG
triplet serves as the initiator codon in approximately 95% of mRNAs.
However, initiation at downstream AUG codons occurs in some specific
circumstances. When the upstream AUG codon is followed shortly by an
in-frame terminator codon, ribosomes can reinitiate translation at the
next AUG codon downstream. When there are fewer than 10 nucleotides
between the cap and the first AUG codon, ribosomes may initiate at both
the first and the second AUG codons. The other mechanism by which
ribosomes can reach an internal AUG codon is termed "leaky
scanning." This phenomenon occurs when the 5'-proximal AUG codon lies
in an unfavorable context for initiation. Manipulating sequences in the
vicinity of the AUG initiator codon and analyzing naturally occurring
viral bifunctional mRNAs suggest that the optimal context for
initiation is CCACCAUGG or CCGCCAUGG. The purine in position Our immunohistochemical analysis revealed that T lymphocytes in thymus and spleen, as well as bronchial epithelial and salivary duct cells, could be sources of the biologically active, full-length limitin protein. With the exception of the kidney, this tissue distribution closely accords with the results of RT-PCR. The apparent absence of protein in an organ that contains mRNA raises the possibility that in addition to transcriptional regulation, protein synthesis can be controlled at the translation or posttranslational steps. Alternatively, these results might be attributable to differences in sensitivities between assays. Selective staining of the thymic medulla as well as RT-PCR analysis of enriched cell suspensions suggest that mature T cells in central and peripheral lymphoid tissues constitutively produce limitin under normal, steady-state conditions. This raises provocative questions about the function of this cytokine in healthy animals. Limitin is a potent inhibitor of B lymphopoiesis, capable of arresting the process from a very early stage in bone marrow.11,15 B cells are not normally made from stem cells in other tissues and it is possible that limitin contributes to an unfavorable environment. It has recently been shown that the thymus is capable of supporting B lymphopoiesis but is normally prevented from doing so through signals delivered via the Notch/Notch ligand family of molecules.38-40 An additional contribution of limitin is possible. Additional roles for this cytokine in lymphoid tissues might include the surveillance of B-lineage malignancies. Constitutive limitin expression in bronchial epithelial and salivary duct cells raises the possibility of other specialized functions in those restricted areas. The 3-dimensional crystal structure of murine IFN- Heparan-sulfated proteoglycans are major components of extracellular
matrix.2 Glycosaminoglycan recognition is a feature of
certain cell adhesion molecules such as CD31.44
Furthermore, this modification is essential for responsiveness of cells
to basic fibroblast growth factor.45 Heparan-sulfated
proteins can influence the differentiation of hematopoietic
cells,46 in part because of their ability to immobilize
cytokines such as granulocyte-macrophage colony-stimulating factor,
IL-3, basic fibroblast growth factor, IL-7, and
IFN-
Submitted January 16, 2002; accepted June 13, 2002.
Prepublished online as Blood First Edition Paper, June 28, 2002; DOI 10.1182/blood-2002-01-0045.
Supported in part by grants from the Japan Research Foundation for Clinical Pharmacology, the Foundation for Research on Leukemia, the Ministry of Education, Science and Culture of Japan, and the Japan Society for Promotion of Science as well as by grants AI-33084 and AI-20069 from National Institutes of Health.
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: Kenji Oritani, Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail: oritani{at}imed2.med.osaka-u.ac.jp.
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© 2003 by The American Society of Hematology.
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  R. Muromoto, M. Ishida, K. Sugiyama, Y. Sekine, K. Oritani, K. Shimoda, and T. Matsuda Sumoylation of Daxx Regulates IFN-Induced Growth Suppression of B Lymphocytes and the Hormone Receptor-Mediated Transactivation J. Immunol., July 15, 2006; 177(2): 1160 - 1170. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
.
5 M 2-mercaptethanol in the presence
of 1 ng/mL IL-7 (R & D Systems, Minneapolis, MN).
) or
without (
) glycosidase F treatment for 2 days. Viable cell numbers
were evaluated with a hemocytometer. Similar results were obtained in 3 independent experiments. (C) Recombinant limitin was subjected to
Western blot probed with antilimitin under the indicated conditions.
(D) Recombinant limitin was immunoprecipitated with heparin-Sephalose
in the presence of excess of chondroitin sulfate or heparin. Each
precipitate was subjected to Western blot probed with antilimitin.
Similar results were obtained in 3 independent experiments.
3 (3 nucleotides upstream from the AUG codon) and the G in position +4
(immediately following the AUG codon) are particularly important. In
general, when position