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The Journal of Immunology, 1999, 163: 760-765.
Copyright © 1999 by The American Association of Immunologists

M1204, a Novel 2',5' Oligoadenylate Synthetase with a Ubiquitin-Like Extension, Is Induced During Maturation of Murine Dendritic Cells1 ,2

Martin Tiefenthaler3,*, Rainer Marksteiner3,{dagger}, Susanne Neyer*, Franz Koch*, Susanne Hofer*, Gerold Schuler{ddagger}, Michel Nussenzweig§, Rainer Schneider{dagger} and Christine Heufler4,*

* Department of Dermatology and {dagger} Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria; {ddagger} Department of Dermatology, University of Nürnberg-Erlangen, Erlangen, Germany; and § Laboratory of Molecular Immunology, Rockefeller University, New York, NY 10021


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A novel molecule expressed by spleen dendritic cells (DC) was isolated using a subtractive hybridization approach. The full-length M1204 clone has 3063 bp, with 1415 bp spanning a single open reading frame, coding for a protein of a predicted size of about 50 kDa. This sequence has strong homology to 2', 5' oligoadenylate synthetase and contains a ubiquitin-like domain. In Northern blot analyses the mRNA is strongly expressed in spleen DC, whereas, in bone marrow-derived DC, the amount of mRNA increases during the maturation process. None of the other leukocytes nor several hemopoietic cell lines tested express this mRNA, but clear expression occurs in many organs, the highest levels being in thymus, lung, and bone marrow. In situ hybridization, combined with immunocytochemical staining of tissue sections of lung and spleen, shows colocalization of M1204 with the 2A1 and NLDC DC markers. In Western blot experiments, an antiserum raised against the recombinant M1204 recognizes a single band in bone marrow-derived DC and in the lung. The expressed oligoadenylate synthetase domain is active in synthesizing 2',5' diadenylate, which by itself may inhibit viral protein synthesis and may also function as a substrate for 2',5' oligoadenylate synthetase. Since the oligoadenylate/RNase L system provides early protection against virus infection, we hypothesize that M1204 prevents virus-induced cell death in DC.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC)5 are bone marrow-derived APCs specialized to stimulate primary cellular immune responses (1, 2). DC are widely distributed in many tissues and organs, where they reside as immature DC, capturing and processing Ag into immunogenic peptides that are then displayed on MHC molecules on the cell surface. To acquire their function as stimulators of primary T-dependent immune responses, DC undergo a maturation process, including morphological changes, up-regulation of costimulatory molecules, and migration from the peripheral tissues via the bloodstream or lymphatic vessels to T cell areas of the secondary lymphatic organs. Mature DC there perform their function to prime naive T cells and cell-mediated immunity (for a review see Refs. 3 and 4). DC not only are involved in T cell stimulation but also are important in regulating B cell growth and Ig secretion (5, 6, 7), as well as in establishing and maintaining central (8, 9, 10) and peripheral (11, 12, 13) tolerance. Increasing attention is given to the antiviral status of DC, since the cells can be crucial for specific antiviral immune response (14, 15, 16, 17, 18, 19). With influenza, DC are able to control virus replication to a level that allows for Ag presentation but not the induction of apoptosis, as opposed to macrophages (20). Nevertheless, the molecular events of DC development and function are still only partially known.

We have applied a subtractive hybridization approach, subtracting a murine cDNA prepared from spleen DC with cDNA derived from the macrophage-like cell line J774. We present here a novel cDNA encoding a protein with homologies to 2',5' oligoadenylate synthetase and ubiquitin. Proteins of both families are involved in immune responses. 2',5' Oligoadenylate synthetase is part of a system activating RNase L, which is an important unspecific antiviral immune response and is also thought to play a role in the control of cell growth and differentiation (21). Ubiquitin-like proteins can function to modify proteins during immunologic processes, including Ig secretion and T cell activation (22, 23, 24, 25, 26, 27, 28).


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Specific pathogen-free C57BL/6 mice of both sexes were purchased from Charles River (Sulzfeld, Germany) and used at 6–8 wk of age.

Media and reagents

Culture medium was RPMI 1640, supplemented with 10% FCS, gentamicin, and 2-ME.

Dendritic cells

A standard procedure was used for preparation of spleen DCs (29, 30).

Bone marrow-derived DC

Bone marrow-derived DC (BMDC) were grown from precursors to full maturation in 200 U/ml GM-CSF for 8 days as described (31) or were harvested on day 6 as immature DC, using 1-g sedimentation through RPMI 1640, supplemented with 50% FCS, to separate them from the more mature DC.

RNA preparation

total RNA was isolated from all cell types using the Trizol reagent (cat. No. 15596; Life Technologies, Rockville, MD). Organs were homogenized in Trizol using a Polytron T 3000 (Kinematica, Littau, Switzerland).

Construction of the subtracted library

We prepared a cDNA library from both murine spleen DC and the J774 cell line in the Lambda Zap II vector according to the manufacturer’s protocol (cat. no. 236612; Stratagene, La Jolla, CA) and grew them as plasmid pools. The J774 pool was PstI-HindIII cut and biotinylated with a photoactivatable Biotin plus Spacer (Super XX PAB, cat. no. 5030-1; Clontech, Palo Alto, CA) (32) according to the manufacturer’s protocol. The DC pool was EcoRI cut and subtracted with biotinylated vector twice before both cut plasmid pools were mixed for a high stringency hybridization three times using fresh biotinylated J774 plasmid pool each time. The conditions for hybridization were adapted from Sive and John (33). Briefly, 5 µg of vector-subtracted inserts from the DC library were mixed with 100 µg of biotinylated J774 restriction-cut plasmid pool, boiled for 3 min, put on dry ice, and dried in a speedvac centrifuge (Uniequip, Munich, Germany). The pellet was resuspended in 50 µl subtraction buffer (50 mM HEPES (pH 7.6), 0.2% SDS, 2 mM EDTA, and 500 mM NaCl) overlaid with 150 µl mineral oil and kept for 4 days at 65°C. After each round of hybridization, streptavidin was added, and proteinated DNA was removed by phenol extraction. The free nucleic acids consist mainly of DC cDNA.

After completion of three rounds of subtraction, the remaining DC cDNA, enriched for cDNA expressed by DC and not J774 cells, was directly ligated into the Lambda Zap II vector (using the manufacturer’s protocol), thereby obtaining a subtracted library of about 20,000 primary clones. The library was amplified before being analyzed.

cDNA library screening

To screen the library, we first released the inserts by EcoRI digestion and performed Southern blots using as probes the inserts that were visible on ethidium bromide-stained agarose gels to eliminate multiple clones. We discarded all clones with inserts smaller than 100 bp and used the remaining to hybridize lifts of the original DC cDNA library. Clones that were present at a frequency higher than 1 in 100,000 were further characterized by partial sequencing and homology studies. Northern blots were then performed to verify the differential expression of the clones. Hybridization was under standard conditions.

DNA sequencing

DNA sequencing was performed using the chain termination method first described by Sanger et al.(34), using the Sequenase (version 2.0) DNA sequencing kit (cat. no. 70770; Amersham, Amersham, Netherlands). As templates, we used the Bluescript plasmids released from the Lambda Zap II according to the manufacturer’s protocol. Sequence homology studies were performed using the basic local alignment search tool (BLAST) server: blast@ncbi.nlm.nih.gov (35, 36)

Northern blotting

Northern blotting was performed using standard techniques (37).

Combined immunocytochemistry and in situ hybridization

Cryocut sections (10 µm) were prepared and either used immediately or stored desiccated at -80°C and thawed at room temperature before use. The sections were fixed by incubating in 4% paraformaldehyde in PBS for 20 min and washed in PBS for 30 min and in diethyl pyrocarbonate (DEPC)-treated water for 2 min.

Immunocytochemistry was performed first with the following rat mAbs: 2A1, specific for mature DC and B cells (31), and NLDC145, specific for DC and epithelial cells (38), from the American Type Culture Collection (ATCC, Manassas, VA). Ab binding was visualized using biotinylated species-specific sheep anti-rat Ig (Amersham), followed by a streptavidin-Cy 3 conjugate (Sigma, St. Louis, MO). Before continuing with the in situ hybridization, the slides were fixed once more exactly as described. For in situ hybridization, a PCR product spanning nucleotides 764 to 1038 of the M1204 cDNA was prepared and cloned into the pGem T vector (Promega, Madison, WI). The orientation of the insert was determined by partial sequencing. Digoxigenin-labeled RNA was prepared using the Dig-RNA labeling kit (Boehringer Mannheim, Mannheim, Germany). Briefly, 1 µg of SacII- or PstI-cut plasmid was used to prepare sense and antisense digoxigenin-labeled RNA, by reverse transcription with T7 or Sp6 RNA polymerase, respectively, according to the manufacturer’s protocol. Relative concentration of the probes was determined by dot blots. Serial dilutions of the probes were applied to nylon membranes (Genescreen; DuPont-NEN, Mechelen, Belgium), UV light fixed, and incubated with a peroxidase-conjugated anti-digoxigenin Ab (Boehringer Mannheim) that was detected using the ECL system (Amersham). For prehybridization, 200 µl of hybridization solution (50% formamide, 2.5% blocking solution (Boehringer Mannheim), 5x SSC) was applied to the slide and removed after incubation for 1 h at 42°C. The digoxigenin-labeled RNA probe (100 µl), diluted to 100 ng/ml in hybridization solution, was applied to the slides. Incubation was at 42°C overnight.

Stringent washes were at room temperature for 30 min in 2x SSC, followed by 2 x 15 min at 42°C in 2x SSC/50% formamide, and 15 min at 42°C in 0.1x SSC.

For detection of the digoxigenin-labeled probe, we used FITC-labeled {alpha}Dig F(ab) fragment according to the instruction manual (Boehringer Mannheim).

Expression of M1204 on the protein level

Using the full-length clone of M1204 isolated from the DC cDNA library as a template, we amplified the coding region of M1204, using Vent polymerase (cat. No. 254; New England Biolabs, Beverly, MA) and 5[prime-GGAATTCCATATGGACCCGTTCCCCGAC-3' as sense primer and 5'-ACGCGTGACGTCGTAAGGCCTCCAGC-3' as antisense primer. The amplified cDNA was verified by sequencing and introduced into the NdeI and the SalI sites of the bacterial expression vector pRSET5BHis. The fusion protein contained six histidine residues at the C terminus and was controlled by the T7 polymerase. The insert-containing vector was introduced into Bl21 cells by electroporation under standard conditions. Protein synthesis was induced by the addition of 0.5 mM isopropyl ß-D-thiogalactoside (IPTG). Similarly, we amplified the 1.5 ubiquitin-like domains of M1204 with 5'-TCGGAATTCGTTCAGGTGAGAGTG-3' as sense primer and 5'-ACGCGTCGACCTACGTAAGGCCTCC-3' as antisense primer. The amplified cDNA was introduced into the EcoRI annd SalI sites of the bacterial expression vector pMal (New England BioLabs). The insert-containing vector was introduced into JM109 cells under the conditions above described.

Purification of the recombinant M1204 protein

An overnight culture of transformed bacteria (500 ml) was pelleted and resuspended in 30 ml 20% sucrose, 0.8 mg/ml lysozyme, and 0.225 M Tris/HCl (pH 8) and kept on ice for 30 min and solubilized with 9 ml of buffer A (2.5% Brij 58 in 10 mM Tris/Hcl (pH 7.2)), freezing in liquid nitrogen and thawing at room temperature twice. After 20 min incubation on ice, 2-ME was added to a final concentration of 5 mM, and NaCl was added to a final concentration of 0.15 M. The lysate was spun at 40,000 x g. The supernatant and the pellet were used for the purification of the protein under nondenaturing and denaturing conditions, respectively. To the supernatant, 0.5 ml Ni2+-NTA-agarose (Qiagen, Hilden, Germany), equilibrated in buffer B (7.5% sucrose, 0.3% Brij 58, 5 mM 2 ME, 150 mM NaCl, and 75 mM Tris/HCl (pH 8)), was added and mixed for 1 h at 4°C. The resin was packed into a column and washed with 8 ml buffer B, 8 ml buffer B/500 mM NaCl, and 8 ml buffer B. The protein was eluted with buffer B/300 mM imidazole in fractions of 0.4 ml each. Fifteen microliters of each fraction was analyzed by PAGE. The fraction containing the most protein was dialyzed twice for 1 h against 20 mM Mg acetate and 20 mM Tris/HCl (pH 7.5). The protein solution was kept at -80°C. From the pellet, the protein was purified under denaturing conditions, using the Ni2+-NTA-agarose (Qiagen) and the protocol supplied by the manufacturer.

Preparation of Ag for immunization

The protein purified under denaturing conditions was subjected to SDS PAGE at 100 µg/lane. After Coomassie blue staining, the protein-containing bands were cut from the gel and used for immunization of rabbits (conducted by Eurogentec, Seraing, Belgium).

Purification of anti-M1204 Abs

The recombinant protein resembling the 11/2 ubiquitin-like domains of M1204 was purified using the maltose binding system according to the manufacturers protocol, dialyzed against PBS for 2 h at 4°C, and bound to Affi-Gel 10 (Bio-Rad Laboratories, Hercules, Ca) as described by the manufacturer. The protein-coupled resin was packed into a column, and 40 ml of the serum from the final bleed, diluted with 120 ml PBS, was applied to the column. After washing with PBS, the Abs bound to the 11/2 ubiquitin domains were eluted with 100 mM Glycine HCl (pH 2.7) into tubes containing 1 M Tris-HCl (pH 8). Ab-containing fractions were pooled and stored frozen.

Solid phase immunoisolation technique and Western blotting

Immature (day 6) and mature (day 8) BMDC (107) and one whole lung were homogenized with an Ultra-Turrax T25 (IKA, Staufen, Germany) for 10 sec in 1 ml ice cold PBS and treated with ultrasound for 10 sec. A slightly modified solid phase immunoisolation technique (SPIT) (39, 40) was performed using Nunc Maxisorp F Microtiterplates (Nunc, Roskilde, Denmark) coated overnight at 4°C with the Ab eluted from the affinity column in 100 mM carbonate buffer (pH 9.6). Plates were washed with PBS, and 200 µl of cell lysate were incubated for 1 h at room temperature. This was repeated 4 times with fresh lysate each time. After washing again with PBS, the bound protein was eluted with 40 µl PBS containing 1% SDS. Twenty microliters of the sample was subjected to SDS PAGE and electroblotting. The blot was incubated with the affinity-purified antiserum raised to M1204 at a dilution of 1:200 or the whole serum at a dilution of 1:1000. Detection was by anti-rabbit IgG alkaline phosphatase conjugate (Boehringer Mannheim).

Oligoadenylate synthetase assay

Protein (0.5 µg) was incubated with 2 mM ATP, 20 mM Tris/HCl (pH 7.5), 20 mM magnesium acetate, 2 µCi [{alpha}-32P]ATP, and 50 µg/ml poly(I)-poly(C) for 2–18 h at 30°C. The enzyme was inactivated for 5 min at 95°C, and the sample was spun for 10 min at 13,000 x g. Eight microliters was transferred to a fresh tube and treated with 1 U/µl calf alkaline phosphatase (Boehringer Mannheim) in 1 µl 10x buffer for 3 h at 37°C. Two microliters of the sample was applied to a polyethylenamine thin layer chromatography plate and run in 750 mM KH2PO4 (pH 3.5) or 1 M acetic acid. The plates were dried and exposed to x-ray film overnight.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of a novel molecule with homologies to oligoadenylate synthetase and ubiquitin

One of the clones isolated from the subtracted library was M1204. As shown in Fig. 1GoA, the full-length clone was 3065 bp long and had a single open reading frame spanning from nt 720 to 2135, with a methionine codon situated in a consensus Kozac sequence (41) and a polyadenylation site starting at nt 3038.



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  		FIGURE 1. Sequence analyses. A, The 3665-bp clone of M1204 with the deduced protein sequence is shown. The start codon and the polyadenylation sequence are underlined. B, Alignment of M1204 to murine oligoadenylate synthetase and murine ubiquitin. The full sequence of M1204 is aligned to the homologous parts of murine 2',5' oligoadenylate synthetase (MOAs) and murine ubiquitin (Mubi). The consensus sequence is positioned in between, and identical residues are indicated by the corresponding letter. +, Indicates conserved charged or hydrophobic residues.

 
The deduced protein consisted of 473 aa with a predicted molecular mass of about 54 kDa. Sequence comparison to databanks showed little homology to known sequences, except for a stretch (aa 6–337) with homology to 2',5' oligoadenylate synthetase and another stretch at the 3' end of the coding region (aa 408–466) with a high similarity to ubiquitin (Fig. 1GoB).

M1204 mRNA is highly expressed in spleen DC cells and up-regulated during maturation of BMDC

The pattern of cell and tissue distribution as studied by Northern blotting (Fig. 2Go) showed strong expression in DC, but not in other leukocytes and several hemopoietic cell lines, including the following: peritoneal macrophages, the monocyte/macrophage-like cell line J774, the mastocytoma cell line P815, the B cell line A20, and the T cell line EL4. Varying levels of expression could be detected in RNA derived from different organs. Strong expression was seen in bone marrow, lung, thymus, and brain; far less in spleen, heart, and kidney; and little if any expression in liver.



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  		FIGURE 2. Northern blot analyses. A, Three micrograms of poly(A)+ mRNA (lanes 1–5) or 10 µg (lanes 6 to 15) or 15 µg (lanes 16 and 17) of total RNA were loaded as indicated on top of each lane. Hybridization was with a 1200-bp insert of M1204 labeled with [32P]dCTP by random priming. Exposure to x-ray film was overnight at -80°C with enhancer screens. The positions of the 18 S and 28 S ribosomal RNA are shown on the left. B, As a control, GAPDH probes were applied to the same blots.

 
Expression of M1204 mRNA in BMDC, harvested at two different stages of maturation (day 6, immature DC; day 8, mature DC), increased with time of culture, i.e., with maturation.

M1204 mRNA is expressed in NLDC145- and 2A1-positive cells

To identify cells that expressed M1204, we performed in situ hybridization combined with immunocytochemistry (Fig. 3Go).



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  		FIGURE 3. In situ hybridization and immunocytochemistry on tissue sections. In situ hybridization is shown in green (FITC), and the immunocytochemistry performed with 2A1 or NLDC145 is in red (Cy 3). Double positive cells appear yellow. A, Lung section stained with NLDC145: A1, epithelial lining of a bronchiole; A2, sense control. B, Spleen sections stained with 2A1: B1, red and white pulp of spleen (50-fold); B2, detail (100-fold) of B1 showing the T cell area; B3, sense control.

 
As shown in Fig. 3Go, row A, in the lung, the epithelium lining the airways stains red for NLDC145, and below is a network of cells expressing the M1204 gene.

In spleen sections, where Northern blot analyses gave only weak signals (Fig. 3Go, row B), B cell areas had 2A1-positive B cells that did not stain for M1204 (row B, column 1). In the T cell area, we found double positive DC (row B, column 2).

The data suggest that M1204 mRNA is expressed in DC in both organs tested.

Expression of M1204 on the protein level

For the preparation of recombinant M1204 protein, we used six different expression systems, with only one yielding recombinant protein (Fig. 4Go), probably due to toxic effects on bacteria. The protein was mainly produced in inclusion bodies, and only minute amounts could be purified under nondenaturing conditions, to be used for functional assays. The antiserum raised against the denatured M1204 protein was affinity purified using the 1.5 ubiquitin domains of M1204 as the affinity reagent. As shown in Fig. 5Go, from lysates of mature and immature BMDC and of lung, the M1204 protein was isolated by solid phase immunoisolation as a single band at the estimated size of about 55 kDa.



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  		FIGURE 4. Expression of the recombinant M1204 protein. A, Coomassie blue-stained polyacrylamide gel. Crude extract of recombinant Escherichia coli before induction (-) and after induction with 0.5 M isopropyl ß-D-thiogalactoside (IPTG) (+). The induced recombinant protein is marked on the right side (<), and the 50-kDa size marker is indicated on the left (-). B, Western blot performed with nickel-nitrilotriacetic acid (Ni-NTA) alkaline phosphatase conjugate (Qiagen). The second protein appearing on the Western blot is an unrelated histidine-rich, E. coli-derived protein.

 


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  		FIGURE 5. Detection of the M1204 protein in cell lysates. Western blot performed with affinity-purified anti-M1204 serum and anti-rabbit IgG alkaline phosphatase conjugate on M1204 isolated from lysates of 107 day 6 (lane 1) and day 8 (lane 2) BMDC and whole lung (lane 3) by SPIT. Lane 4 contains eluted products from the Ab-coated plate only. The predicted size of M1204 (55 kDa) is marked on the right side (<), and the 50-kDa size marker is on the left (-).

 
M1204 synthesizes 2',5' diadenylate

With regard to the putative functional role of the molecule, we have focused on the oligoadenylate synthetase domain. We used the recombinant protein in oligoadenylate synthetase assays. Only the protein purified under nondenaturing conditions was active in synthesizing oligoadenylate (Fig. 6GoA). We found that the product synthesized by the recombinant M1204 was independent of the presence of dsRNA. To investigate the extent of polymerization of the above product, thin layer chromatography was additionally performed with 1 M acetic acid (42) (Fig. 6GoB), comparing the oligoadenylate synthesized by M1204 with size markers. We found the products of M1204 in the oligoadenylate synthetase assay to be exclusively diadenylates.



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  		FIGURE 6. Oligoadenylate synthetase assay. Autoradiogramm of the products obtained after incubation of [32P]ATP with M1204 protein. A, Thin layer chromatography was performed with 0.75 M KH2PO4. Lane 1, No protein added; lane 2, with protein and addition of dsRNA; lane 3, with protein, without addition of dsRNA. Positions of inorganic phosphate (Pi) and 2',5' diadenylate (2'-5' A) are marked. B, Thin layer chromatography performed with 1 M acetic acid. Lane 1, No protein added; lane 2, with protein and addition of dsRNA; lane 3, with protein, without addition of dsRNA. Positions of inorganic phosphate (Pi), 2', 5' diadenylate (2'-5' A), and size marker (SM; cat no. A6400, Sigma) are marked.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
From a subtractive hybridization we have isolated a novel molecule that is highly expressed in mature mouse DC. The colocalization of M1204 mRNA with the DC Ag 2A1 in the T cell area of the spleen identifies these cells as DC (interdigitating cells) (21). In the lung, M1204 is expressed by a layer of DC at the base of the airway epithelium. In mice, these cells are not stained with MHC II, but their location is identical to that discussed by Holt and colleagues for rat and human airway DC (43, 44). For further studies, we will use the antiserum for double immune staining.

Two domains of M1204 showed homology to 2',5' oligoadenylate synthetase and to ubiquitin. 2',5' Oligoadenylate synthetase is part of a system that restricts virus spreading by activating RNase L, an RNase that can selectively degrade viral RNA (45). Therefore, we expressed M1204 as a recombinant protein and used it in oligoadenylate synthetase assays. M1204 was active in synthesizing 2',5' diadenylate but not higher oligomers. However the homology of M1204 to 2',5' oligoadenylate synthetases is not as high as between the different forms of 2',5' oligoadenylate synthetases (46). In contrast to 2',5' oligoadenylate synthetases, M1204 exhibited its activity both with and without the presence of dsRNA. Diadenylates have been shown to have an independent antiviral activity of unknown molecular mechanism (21, 47). Although diadenylate itself does not activate RNase L, it could function as a substrate for 2',5' oligoadenylate synthetases (48) to accelerate the production of higher polymers that can activate RNase L.

The oligoadenylate synthetase/RNase L system is usually activated by IFN treatment in the presence of dsRNA and is part of an antiviral immune response. The finding that DC during the maturation process up-regulate a molecule with antiviral activity, or the capacity to constitutively provide the substrate for faster activation of RNase L, may implicate M1204 in protecting the DC from virus infection via Ag uptake and processing, thereby preventing extensive viral replication and cell damage. DC exhibit distinctive features when infected with influenza virus. The replication rate is about 10-fold lower than in macrophages, and DC, unlike macrophages, do not undergo rapid apoptosis (20). Since macrophages also in situ (macrophages in red pulp and lung airways) do not normally express M1204, a protective function of M1204 during Ag uptake by DC could account for the observed differences between DC and macrophages infected with influenza virus. On the protein level, we show expression of M1204 in mature and immature BMDC as well as in the lung. SPIT used to isolate the M1204 protein from the lysates does not allow for quantitative analyses; nevertheless, in accordance with its putative function as an early protection to virus infection, the M1204 protein is expressed already in immature BMDC. We will attempt to evaluate the role of M1204 on the susceptibility/resistance of cells to influenza virus infection in an ongoing study.

2', 5' Oligoadenylate synthetase is also thought to be involved in the general mechanisms of cell growth regulation, especially in the antiproliferative mechanisms of resting cells (49). It is therefore possible that M1204 is involved in down-regulating proliferation of DC.

For the ubiquitin-like domain, we assume a potential protein interaction site. However, when we expressed the ubiquitin-like domain and used the recombinant protein for binding studies, we could not identify any binding partner. Addition of the ubiquitin-like domain of M1204 to the oligoadenylate synthetase assays did not interfere with the enzyme activity (data not shown), implicating an independent function of this domain.


   Acknowledgments
 
We thank Dr. Peter Fritsch, Chairman of the Dept. of Dermatology, for his continued support and Dr. R. M. Steinman for carefully reading the manuscript.


   Footnotes
 
1 This work was supported by Austrian Science Fund (FWF) Projects P11522-Med and P10897-Med, the Austrian National Bank (Jubiläumsfond, Project 5891), and Deutsche Forschungsgemeinschaft Project 1186/1-2. Back

2 The sequence data reported here have been submitted to the GenBank database under accession No. AF068835. Back

3 M.T. and R.M. have contributed equally to this paper. Back

4 Address correspondence and reprint requests to Dr. Christine Heufler, Department of Dermatology, University of Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austria. E-mail address: Back

5 Abbreviations used in this paper: DC, dendritic cells; BMDC, bone marrow-derived DC; SPIT, solid phase immunoisolation technique. Back

Received for publication December 4, 1998. Accepted for publication April 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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