Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M302854200 on May 9, 2003

J. Biol. Chem., Vol. 278, Issue 33, 30889-30895, August 15, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/33/30889    most recent
M302854200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Potolicchio, I.
Right arrow Articles by Strominger, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Potolicchio, I.
Right arrow Articles by Strominger, J. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Molecular Interaction and Enzymatic Activity of Macrophage Migration Inhibitory Factor with Immunorelevant Peptides*

Ilaria Potolicchio {ddagger} §, Laura Santambrogio § and Jack L. Strominger {ddagger} 

From the {ddagger}Department of Molecular Cell Biology, Harvard University, Cambridge, Massachusetts 02138 and §Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115

Received for publication, March 20, 2003 , and in revised form, May 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Disulfide reduction is an important step in antigen processing for HLA class II restricted T cell responses. Migration inhibitory factor (MIF) is a member of the thioredoxin family and has been classically defined as a cytokine. Using enzyme-linked immunosorbent assay and CD analysis, here we describe the binding to MIF of two peptides, hepatitis B surface antigen (HBsAg) and insulin B (InsB) with high affinity for HLA class II allo-types, HLA-DP2 and HLA-DQ8, respectively. At neutral pH, cysteinylated InsB was a substrate for MIF thiol reductase activity, as assessed by mass spectroscopy/electrospray analysis. Finally, a biologically active form of MIF co-immunopurified with mature forms of HLA DP2/15, and a peptide derived from the HLA-DP {beta}1 helix could be used for affinity purification of MIF. The possibility that MIF participates in class II antigen presentation and/or as a chaperone is discussed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The macrophage migration inhibitory factor (MIF)1 is a multifunctional protein involved in several inflammatory disorders (1, 2), such as inflammatory lung diseases (3), septic shock (4), chronic colitis (5), and some autoimmune diseases, e.g. rheumatoid arthritis (6). MIF is widely expressed in different cell types but is produced mostly by activated macrophages and lymphocytes during inflammatory processes (7). The full extent of its physiological role is not completely defined, although MIF has been implicated in several biological activities. First, MIF can function as a pro-inflammatory protein, as assessed by its inhibitory effect on dexamethasone-mediated TNF-{alpha} production (2) and its ability to sustain CD3-mediated T cell proliferation (8) and pro-inflammatory cytokine secretion (9). MIF also possesses enzymatic activities as a tautomerase/isomerase (10) and thiol oxidoreductase (11). However, the natural substrates for MIF catalytic activity are unknown even though its enzymatic activity has been characterized in detail using insulin and L-DOPA as models (11, 12).

X-ray crystallographic analysis of both human and rat recombinant MIF molecules showed a homotrimeric structure (13, 14). However, MIF is predominantly expressed as the monomer (44%) and dimer (33%), whereas only a smaller fraction (23%) is assembled to form a trimer (15, 16). Native MIF presumably possesses all three configurations of its recombinant forms. However, the relationship, if any, between configuration and biological activity is still to be defined.

Antigen-presenting cells are able to internalize extracellular proteins within the acidic endosomal and lysosomal compartment to generate peptides for HLA class II loading. Antigen processing consists of protein denaturation and fragmentation. Several proteases have been described to be part of this system (17). Proteins that contain disulfide bonds require an additional processing step, the reduction of the cysteine residues to produce antigenic peptides (18). Notably, modification of cysteine residues has been recently shown to regulate T cell responses to HLA class II-restricted epitopes (19–21). Within the endosomal pathway the {gamma}-interferon-inducible lysosomal thiol reductase, the activity of which is restricted to acidic pH, has been found associated with antigen processing using reduction of cysteinylated peptides as the assay (22).

Antigenic peptides can also be generated extracellularly by secretion of several proteases from professional antigen-presenting cells or stressed cells, such as virally infected and tumor cells (23–25). Additionally exogenous antigens or already unfolded proteins can bind directly to surface HLA class II molecule for T cell presentation (26, 27).

Here we describe the MIF peptide binding capacity and, more importantly, MIF thiol reductase activity at neutral pH on disulfide bonds in oxidized peptides. Because MIF is a secreted protein, particularly during inflammation, its thiol reductase activity could be associated with peptide modification in the extracellular milieu. Moreover, because MIF co-precipitated with HLA-DP2/15 molecules, a further role for MIF in HLA class II antigen presentation is suggested.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Synthetic Peptides—The following peptides were synthesized by Research Genetics (Waltham, MA): residues {beta} 63–77 (KDILEEERAVPDRMA) of HLA-DP2 molecule; residues 306 –318 (PKYVKQNTLKLAT) of influenza virus hemagglutinin (HA); residues 14 –33 (VLQAGFFLLTRILTIPQSLD) of hepatitis B surface antigen (HBsAg); residues 85–99 (ENPVVHFFKNIVTPR) of myelin basic protein and residues 9 –23 (SHLVEALYLVCGERG) from the insulin B chain (InsB).

Cloning and Bacterial Expression of Human MIF—MIF was amplified from the cDNA of a human B cell line, NP-1, and cloned in an isopropyl-1-thio-{beta}-D-galactopyranoside-inducible expression vector (PGMT-7). PCR primers were designed as previously described with NdeI (5'-GCA TAT ACA TAT GCC GAT GTT CAT CGT A-3') and BamHI (5'-TTG GAT CCT TAG GCG AAG GTG GAG TT-3'). MIF-PGMT-7 was expressed in Escherichia coli BL21-(DE3) pLysS+ cells (Invitrogen). One liter of bacterial cell culture was grown at 37 °C until the optical density at 595 nm reached was 0.7–1 absorbance units, and then the cell culture was incubated with 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside for2hat30 °C. The pellet was resuspended in 5 ml/g BugBaster protein extraction Reagent (Novagen, Madison, WI) for 20–45 min in the presence of DNase and protease inhibitors. Cell debris were removed by centrifugation, and after filtration through 0.45-µm filters, the supernatant was applied to Mono Q column (Amersham Biosciences) and an overnight dialysis at 4 °Cin20mM Tris, pH 7.5, 20 mM NaCl. Because MIF is not retained by Mono Q resin, the flow-through was collected, and MIF was purified by gel filtration chromatography (Superdex 200, Pharmacia).

ELISA and CD Spectrum Analysis—ELISA plates were coated overnight with 50 µM each peptide and then blocked for 1 h in 2% bovine serum albumin, phosphate-buffered saline at room temperature. Two micrograms of recombinant MIF (rMIF) were added and incubated overnight with the peptides. After washing, bound rMIF was detected in DuoSet ELISA (R&D, Minneapolis, MN). For CD spectrum analysis 25 µM rMIF was incubated overnight at room temperature with or without 2.5 µM each peptide. The incubation was performed in HSB (20 mM Hepes, 0.85% NaCl). The next day samples were diluted 10x in water, concentrated with MICRON10 (Millipore, Bedford, MA), and analyzed on a Jasco CD spectrometer. Secondary structure was analyzed on line by using the k2d program (www.emblheidelberg.de/~andrade/k2d).

Thiol Reductase Activity of MIF—Insulin B, residues 9 –23, was oxidized as previously described. Briefly, peptide was incubated with 400 µM Cys-Hank's balanced salt solution for 3 h and then dialyzed for 48 h against 10 mM Hepes, pH 7.0. 0.5 µg of oxidized peptide was incubated for 20 min with increasing amounts of rMIF, 100, 200, and 500 ng in 10 µl of 0.4 mM Cys-10 mM Hepes buffer, pH 7.0. Samples were purified by ZipTip (Millipore) and analyzed by mass spectrometry electrospray. Mass spectrometry electrospray analysis was carried out at the Chemistry Department at Harvard University.

Immunoaffinity Columns—HLA-DRB1*0401 was purified from 10 g of B cell line pellet (PRIESS). HLA-DP2/15 was purified from 20 g of a heterozygous B cell line (NB-1) transformed and previously typed. Pellets were resuspended in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 0.1 mM NaCl, complete EDTA-free protease inhibitory mixture tablets (Roche Applied Science), and either 1% Nonidet P-40 or 0.5% CHAPS. After centrifugation protein extract was filtered through a 0.2-µm filter and put through a series of columns: POROS(r)20 AL-NMS, POROS(r)20 A, POROS(r)20 AL-LB3.1, POROS(r)20 AL-IVD12, and POROS(r)20 AL-B7/21 as previously described (35). After a protocol set up in this laboratory, immunoaffinity columns were washed with solution I (0.1% Nonidet P-40, 50 mM Tris-HCl, pH 8.0), solution II (0.5% CHAPS, 500 mM NaCl, 50 mM Tris, pH 8.8), and solution III (0.1% CHAPS, 2 mM Tris-HCl, pH 8.0). Elution was in 0.1% CHAPS, 50 mM glycine buffer, pH 11.5. Buffer exchange and protein concentration were performed on Centricon 10. Small scale protein purification was obtained using cell lysate from 5 g of B cell line. Native MIF was isolated using the monoclonal antibody anti-human MIF (R&D) conjugated to N-hydroxysuccinimide-activated resin (Amersham Biosciences). Second-step purification was performed using either B7/21 alone or a mix of HLA class II monoclonal antibodies, B7/21 and IVD12, conjugated to N-hydroxysuccinimide-activated column. PRIESS and NB-1 were both analyzed. Proteins were run on 12.5% SDS gels and analyzed by Coomassie Blue staining or Western blot. HLA class II isotypes were stained with a general anti-HLA class II rabbit serum, generously provided by Dr. N. Tanigaki. Human MIF was stained using polyclonal biotinylated antibody specific for human MIF (R&D).

Analysis of the Inhibitory Effect of MIF on Dexamethasone Activity— MIF function was tested as previously described. Briefly, human monocytes were isolated from whole blood by Ficoll density gradient centrifugation and plated at 5 x 106 cells/ml in 24-well plates in RPMI, 10% human AB serum. The monocytes were purified by adherence and incubated for 1 h with dexamethasone (10–9 M) plus 1–10 ng/ml of rMIF (R&D) or 1 ng of human MIF co-purified with HLA-DP2/15 from B7/21 column. LPS (Sigma) was added at the concentration of 0.5 µg/ml. Sixteen hours of conditioned medium was collected, and TNF-{alpha} secretion was quantified by ELISA (DuoSet R&D).

Pulse-Chase and Immunoprecipitation—LB and Bg5 B cells lines were pulsed for 30 min with 1 µCi/ml [35S]methionine (PerkinElmer Life Sciences) and then chased for 1 and 4 h in complete Dulbecco's modified Eagle's medium supplemented with a 10-fold excess of cold methionine and cysteine. Cells were lysed in 1% Nonidet P-40 150 mM NaCl in 50 mM Tris containing a mixture of protease inhibitors (Roche Applied Science) and equivalent amounts of radioactive post-nuclear supernatant used for immunoprecipitation after preclearing with protein A- and G-Sepharose beads (Sigma). The immunoprecipitation was performed using a mouse anti-human MIF mAb (clone 12302.2) (R&D system). Samples were resolved by SDS-PAGE boiled and non-boiled under non-reducing conditions.

Peptide Affinity Column—A HiTrap N-hydroxysuccinimide-activated 1-ml column (Amersham Biosciences) was conjugated with 5 mg of solubilized peptide following the protocol provided by the company. Five grams of a transformed human B cell line were lysed in 25 mM Tris-HCl, 0.5 mM NaCl, 0.5% CHAPS, and complete, EDTA-free, protease inhibitor mixture tablets (Roche Applied Science). The affinity column was loaded overnight at 4 °C with cell lysate and then extensively washed in lysis buffer. Elution was completed with 100 mM glycine buffer, pH 3. Fractions corresponding to the elution peak were concentrated on Micron 10 (Millipore) and analyzed by Coomassie staining of 12.5% SDS-PAGE. The whole elution was trypsin-digested and sequenced by matrix-assisted laser desorption ionization mass spectroscopy analysis. Tandem mass spectroscopy analysis was carried out at the Microchemistry Facility at Harvard University.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
MIF Peptide Binding Analysis—Macrophage migration inhibitory factor functions as a tautomerase/isomerase and thiol reductase on small molecule substrates such as L-DOPA and insulin (11, 12). Pro-1 and Cys-57–Cys-60 form the enzymatic catalytic sites. Thioreductase activity has an optimal pH ranging between 7.3 and 9 (11). MIF is a soluble protein mostly secreted extracellularly. Because a number of different molecules with enzymatic properties have been found involved in extracellular protein fragmentation and processing (23–25) the possible interaction between MIF and immunologically relevant peptides was examined. Human rMIF was expressed and purified from E. coli BL21-(DE3) Lys+ cells as previously described (28). Correct protein folding assessed by CD spectrum analysis (see Fig. 2) was identical to that previously published (29). Several well known high affinity HLA class II peptides were selected and analyzed for their ability to interact with rMIF in an ELISA assay (Fig. 1) (30). The highest amount of rMIF was detected in plates coated with HBsAg14–33, a peptide derived from hepatitis B surface antigen, which showed a high binding affinity for HLA-DP2 (31). A fragment of insulin B (InsB9–23) known to bind HLA-DQ8 (32) also showed a significant binding capacity to rMIF, whereas little or no binding was observed for both myelin basic protein residues 85–99 and HA306–318, peptides, known for their high affinity binding to HLA-DR2 and HLA-DR1, respectively (33). Although ELISA is a well established method to determine HLA peptide binding, a better approach to examine protein-protein interaction is analyzing conformational changes by CD spectrum analysis. In agreement with what has been previously reported, the rMIF CD spectrum showed a positive ellipticity at 197 nm and broad negative ellipticity between 205 and 225 nm (Fig. 2) (29). Under the conditions used, the secondary structure obtained with the K2d program was 37% {alpha} helix, 15% {beta} sheet, and 48% random coil. Moreover, changes in the MIF CD spectrum were observed after an overnight incubation with HBsAg14–33 peptide (Fig. 2a). Secondary structure analysis by K2d revealed a consistent increase of {beta} sheets and decrease of random coils, 41 and 31%, respectively. No modifications, either in CD spectrum or in secondary structure, were observed after an overnight incubation with HA306–318 or myelin basic protein residues 85–99 peptides (Fig. 2b). At the concentration used all of the peptides had only a very weak signal that coincided with the noise level (Fig. 2c). These observations suggest that rMIF has a flexible conformation, and major modifications of the secondary structure may occur upon complex formation with specific peptides.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2.
CD spectrum analysis of MIF-peptide complexes Far-UV CD spectroscopy of rMIF (blue) in comparison with rMIF-HBsAg14–33 complexes (magenta) (a), rMIF-HA306–318 complexes (green) (b), and myelin basic protein rMIF-MBP residues 85–99 complexes (red). c, peptide CD signal.

 


View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1.
Analysis of peptide binding MIF. rMIF was added to ELISA plates previously coated with 50 µM HLA class II naturally processed peptides. The amount of bound protein was quantified using a biotinylated polyclonal antibody specific for human MIF followed by horseradish peroxidase-conjugated-Streptavidin. Absorbance was measured at 450 nm. The data shown represent a typical experiment where each sample is assayed in triplicate. The experiment was repeated three times.

 

MIF Thiol Reductase Activity—MIF enzymatic activity includes a thiol reductase function (11). Site-directed mutation analysis pointed to Cys-57 and Cys-60 as part of the MIF enzymatic catalytic site, determined in vitro using molecular models (11). Further analysis was conducted here to investigate whether the MIF thiol reductase activity could function on oxidized peptides. Among the synthetic peptides tested for their binding to rMIF (Fig. 1), InsB9–23, molecular mass 1645.8, possesses a cysteine residue in position 11. Cysteinylation of this residue in Cys-Hank's balanced salt solution changed the molecular mass to 1765, as shown by mass spectrometry electrospray analysis (Fig. 3, a–b). Peaks at 823 and at 883 are double-charged and correspond to m/z of reduced and oxidized peptide, respectively. After 20 min of incubation with rMIF at pH 7.2 a significant amount of InsB9–23 was reduced (Fig. 3c). The ratio peptide reduced/oxidized was dose-dependent as shown in Fig. 3d. Thus, rMIF can function directly to reduce cysteinylated forms of InsB 9–23. As expected, no modifications in the molecular mass of the cysteinylated peptide occurred without adding rMIF (Fig. 3b). Thus, peptides can be substrates of MIF thiol reductase, and as expected, this reaction occurs at neutral pH



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3.
MIF thiol reductase activity. Mass spectrometry electrospray analysis of reduced InsB9–23, m+2 823 (a), oxidized InsB9–23, m+2 882 (b), and oxidized InsB 9–23 after incubation with 200 ng of rMIF (c). d, schematic representation of the dose-dependent reduction of Cys-InsB9–23 by purified rMIF. The ratio of relative peaks, m+2 reduce peptide and m+2 cysteinylated peptide, are indicated.

 

MIF and HLA Class II Association—A possible association between MIF and HLA class II molecules was also assessed. Recently a modified methodology based on high pressure immunoaffinity columns that is suitable for rapid large scale purification of HLA proteins was described (34, 35). This technique was applied to purify HLA-DR (HLA-DRB1*0401) using mAb LB3.1 and HLA DP (HLA-DPB1*0201/*1501) using mAb B7/21 affinity columns. The isolated HLA molecules were analyzed on Western blot using a general HLA class II rabbit antiserum as the detection antibody (Fig. 4a, left). Interestingly, MIF was found associated with HLA-DP2/15 mainly as the dimer, as detected by Western blot analysis, but not with the HLA-DR4 preparation (Fig. 4a, right).



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 4.
Analysis of human MIF interaction with HLA class II molecules. a, Western blot analysis of immunoaffinity-purified HLADR4 and HLA-DP2/15 molecules and Western blot analysis of MIF co-precipitated with HLA class II molecules. MIF monomeric and dimeric forms are indicated. b, quantification of the amount of MIF found in association with HLA-DP2/15 molecules (filled diamonds) and HLA-DR4 (filled circle). Protein standards are indicated as open squares. c, secretion of TNF-{alpha} in untreated PBLs (Unt, lane 1) and in LPSstimulated PBLs (lane 2). The inhibitory effect of dexamethasone (Dex) on TNF-{alpha} secretion in LPS stimulated PBLs in shown in lane 3. The antagonist effect of rMIF (1–10 ng/ml) (lane 4) and human MIF (hMIF, 1 ng/ml) co-isolated with HLA-DP2/15 (lane 5) on TNF-{alpha} secretion in dexamethasone-LPS treated PBLs are shown. Secretion of TNF-{alpha} in PBLs is shown treated with dexamethasone (lane 6), rMIF (lane 7), or human MIF (lane 8) without LPS stimulation. NB, non-boiled.

 

MIF has been characterized by its ability to override the inhibitory effect of dexamethasone on TNF-{alpha} production from LPS-stimulated human peripheral blood mononuclear cells (9). This assay was, therefore, used to assess the biological activity of MIF found in association with HLA-DP2/15. First, sandwich ELISA was performed to detect the MIF concentration in the HLA-DP preparation (Fig. 4b). The HLA-DR preparation was also analyzed, and as expected, no MIF was found associated. As shown in Fig. 4c, MIF that had been co-purified with HLA-DP2/15 as well as purified recombinant MIF partially antagonized the inhibitory effect of dexamethasone on TNF-{alpha} secretion to an extent similar to that previously reported (9).

Pulse-chase experiments were performed to investigate whether MIF could be found associated with HLA DP2/15 at any step of class II trafficking from the endoplasmic reticulum to the cell surface. HLA class II heterodimers were chased up to 18 h; however, MIF could not be identified after precipitation of HLA-DP (data not shown). This result was not surprising since, besides the invariant chain, which associates with all nascent class II {alpha}/{beta} heterodimers, all other proteins known to pair with HLA class II molecules (e.g. HLA-DM, HLA-DO (36), tetraspanin (37, 38), and CD1d (39)) only interact with a small percentage of the class II {alpha}/{beta} complexes. HLA-DP-associated MIF was detected by first immunopurifying with the mAb specific for human MIF (Fig. 5a) followed by a second immunopurification step using mAb B7–21 specific for HLA-DP molecules (Fig. 5b). Interestingly, MIF is associated with peptide-loaded forms of HLA-DP, as indicated by the presence of an SDS stable class II {alpha}/{beta} complex. In these experimental conditions, MIF dimers and monomers were purified using both anti-DP and anti-MIF column (Figs. 4a, 5a, and 6c) and the dimeric form of MIF became monomeric under reducing conditions (Fig. 5a). In pulse-chase experiments on two different B cell lines (LB, Bg5), MIF was primarily observed as a monomer. However, a faint band corresponding to the dimeric form was also evident (Fig. 5c).



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 5.
Native MIF isolation and analysis of HLA class II molecule association. a, MIF was immunoaffinity-purified and the eluted protein was analyzed by Western blot. MIF monomeric and dimeric forms are both visible. b, eluate from the MIF column was re-precipitated with the B7/21 anti-HLA-DP antibody. Samples were analyzed under non-reducing conditions with anti-HLA Class II rabbit serum. To ensure specificity, all the monoclonal and polyclonal Abs used in immuno-affinity purification were tested for cross-reactions. c, pulse-chase analysis of human MIF in two different B cell lines.

 


View larger version (23K):
[in this window]
[in a new window]
  		FIG. 6.
Isolation of native MIF using HLA-DP2 {beta} 63–77) peptide affinity column. Total cell extract from the NB-1 B cell line was loaded on a peptide affinity column conjugated with a peptide spanning amino acids {beta} 63–77 from HLA-DP2. Eluted proteins were analyzed by Coomassie staining of a SDS-PAGE (a), by tandem mass spectroscopy sequence analysis (b), by Western blot analysis using anti-MIF monoclonal antibody (c).

 

Monomeric MIF possess three cysteines, Cys-56, Cys-59, and Cys-80. Because substitution of Cys-59 led to a significant reduction of the dimeric and trimeric forms, it is likely that the same position is involved in intermolecular disulfide bonds, as suggested by experiments shown in Figs. 4a, 5a, and 6c. Whether such an S-S bridge is an in vitro artifact or exists under physiological condition require further investigation.

MIF Can Be Isolated by Column Affinity Using the Synthetic 15-mer DP {beta} 63–77 Found in HLA-DPB1*02—X-Ray crystallographic analysis of HLA-DR molecules identified a floppy region around the break point of the {beta}1 helix important for its structural flexibility (40). The same region may be involved in the HLA-DR4/Hsp70 interaction since a peptide spanning residues {beta} 64–78 has been used to purify Hsp70 (41). Molecular modeling suggests the presence of the same bulge for HLA-DP2 molecules (42). To investigate whether MIF interacts with this acidic stretch of HLA-DPB1, a peptide spanning residues {beta} 63–77 of HLA-DP1*02 was conjugated with N-hydroxysuccinimide-activated Sepharose matrix (Fig. 6a). A column was loaded with crude proteins extracted from a human B cell line (see "Experimental Procedures"), and the eluate was analyzed by separation on SDS-PAGE. Human MIF was the only substance that could be detected by Coomassie Blue staining (Fig. 6a) or by matrix-assisted laser desorption ionization/time of flight analysis (Fig. 6b). The sample was also analyzed by Western blot analysis with MIF antiserum confirming the presence of MIF (Fig. 6c).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
MIF crystal structure analysis showed a homotrimeric structure shaped as a single ring of 15-Å diameter at the open ends, supposedly the binding site for small molecules (13). However, crystallographic analysis and NMR are in discordance with the observation that MIF in physiological conditions exists primarily as a monomer and dimer. Evidence also suggests that dimer or monomer mediate MIF function (7), although the relation between structure and functionality is still ill defined.

MIF tautomerase/isomerase activity relies on Pro-1 that promotes enolization and ketonization (10, 43). Because its natural ligand is unknown, MIF enzymatic properties have been described in detail using molecular models as substrates (44). Among those that have been tested are the physiological molecules L-DOPA and insulin (45). Because insulin would not fit within the channel formed by its trimeric assembly, MIF interaction with substrates such as insulin would probably require a dimeric or monomeric structure. The present report that HLA class II peptides can also be MIF substrates is a novel characteristic.

An additional enzymatic activity reported for MIF is its thiol reductase function (11). The molecular redox center has the conserved sequence motif of the thioredoxin family consisting of CXXC (Cys-57–Ala–Leu–Cys-60) (11). MIF thioreductase activity has been well characterized on small proteins. Here, in addition to defining MIF peptide binding capacity, its thiol reductase activity is also demonstrated to function on a peptide substrate as well, cysteinylated (InsB9–23). Another enzyme of the thioredoxin family was recently reported, {gamma}-interferon-inducible lysosomal thiol reductase. {gamma}-Interferon-inducible lysosomal thiol reductase is constitutively expressed in professional antigen-presenting cells and catalyzes disulfide bond reactions at low pH (46). Interestingly, {gamma}-interferon-inducible lysosomal thiol reductase specifically associates with HLA-DRw52 (47)2 and has been shown to be very important for HLA class II peptide presentation. In fact a cysteine-containing peptide ({kappa}I) can be converted from an "inactive" to an "active" form in inducing T cell proliferation only in the presence of {gamma}-interferon-inducible lysosomal thiol reductase even though both peptide forms are similarly loaded on HLA class II molecules (20). An understanding of the disulfide catalytic activity of MIF on peptide substrates is a new challenging aspect of its biological function. Notably, the reducing property of MIF occurs at neutral pH, different from {gamma}-interferon-inducible lysosomal thiol reductase that functions at lysosomal pH. Thus, either the extracellular environment or the cytoplasm are the possible reaction sites of MIF. Several proteases function extracellularly as cathepsin S and L (17, 48), metalloprotease (MMP-3, MMP-9, and MMP-12) (49, 50), and membrane-associated ectoenzymes (CD13 and CD26) (51). As a result, antigenic determinants can be generated extracellularly for loading onto HLA proteins. Thus, the peptide-editing activity of MIF on cysteine residues is likely part of the extracellular processing machinery. Secretion of thioredoxins has been correlated to the generation of a reducing extracellular environment necessary to sustain T cell activation and proliferation (52). Conversely to an oxidative milieu, reducing environments would not alter the oxidative conditions of antigens after their processing. Thus, the reducing condition required to activate T lymphocytes could also preserve the extracellular "active" or "inactive" state of certain peptides during antigen presentation.

Recently, much interest has surrounded the understanding of the role of soluble "peptide chaperones" during antigen presentation processes. A number of heat shock protein family members (Hsp70, Hsp90, gp96, Hsp110, gpr170) have been shown to interact with a broad range of peptides (53, 54). Historically, Hsp function has been described for class I HLA loading where peptides are transported from the cytosol to the endoplasmic reticulum in complex with Hsps (55). However, some data suggest an intracellular Hsp involvement in HLA class II presentation as well (55). For example, Hsp70 interacts with specific peptide fragments derived from the third hypervariable region of HLA-DRB1*0401 and co-precipitates with HLA-DR4 molecules (56). Similarly, in this report MIF association with mature forms of HLA-DP2/15 molecules is described. All together, these observations, MIF peptide binding capacity and its interaction with HLA-DP molecules, support its possible involvement in HLA class II peptide generation and exchange and, in this sense, its additional function as HLA class II chaperone. HLA-DP is the least expressed and least characterized among HLA class II isotypes (34). Its role in antigen presentation has been established for viral and bacterial antigens and in alloreactivity (57). One of the strongest HLA class II genetic associations with immunological disorders is the presence of a glutamic acid at position HLA-DP {beta}69, which confers susceptibility to metal-induced disorders as chronic beryllium disease and hard metal diseases (58, 59). MIF association with other HLA class II polymorphic variants cannot be excluded at the present time and will be a matter of further investigations.

Because MIF was purified using a peptide affinity column loaded with a fragment of HLA-DP {beta}1 domain-spanning residues 63–77, the same region may also be involved in HLA-DP/MIF interaction. This region extends around the breaking point of the {beta}1 helix that is known to be particularly sensitive to peptide-induced structural modifications (60, 61). Furthermore, the positions {beta}46 and {beta}72 in HLA-DR are anchor residues for Epstein-Barr virus gp42 protein and critical for virus entry (62). Although MIF has been originally classified as a cytokine, neither a receptor nor a high affinity membrane molecule has been identified. Its interaction with HLA class II molecules might represent its mechanism of entry into the cell similarly to the pan CD91 receptor, responsible for gp97, Hsp90, Hsp70, and caloreticulin endocytosis (63).

In conclusion, new aspects of MIF biology have been described in this paper; peptide binding capacity, its function as thiol reductase on peptide substrates, and importantly, its association with HLA-DP molecules. These observations point to new functional aspects of this small protein and raise interesting questions about the role of MIF during inflammatory chronic diseases and autoimmunity.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AI-49524 and CA-47554 (to J. L. S.) and AI-48832 (to L. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence may be addressed. Tel.: 617-495-2733; Fax: 617-496-8351; E-mail: ipotolic{at}mcb.harvard.edu or jlstrom{at}fas.harvard.edu.

1 The abbreviations used are: MIF, migration inhibitory factor; rMIF, recombinant MIF; HA, hemagglutinin; HBsAg, hepatitis B surface antigen; InsB, insulin B; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; LPS, lipopolysaccharide; TNF, tumor necrosis factor; Ab, antibody; Hsp, heat shock protein; ELISA, enzyme-linked immunosorbent assay; PBL, peripheral blood leukocytes. Back

2 P. Cresswell, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Lih Wen Deng for providing purified HLA-DR4 molecules, Nobuyuki Tanigaki for a general HLA class II rabbit anti-serum, and Jonathan Boyson for helpful discussion. A special thanks to Lawrence J. Stern and Peter Cresswell for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bernhagen, J., Calandra, T., and Bucala, R. (1998) J. Mol. Med. 76, 151–161[CrossRef][Medline] [Order article via Infotrieve]
  2. Calandra, T., and Bucala, R. (1997) Crit. Rev. Immunol. 17, 77–88[Medline] [Order article via Infotrieve]
  3. Donnelly, S. C., Haslett, C., Reid, P. T., Grant, I. S., Wallace, W. A., Metz, C. N., Bruce, L. J., and Bucala, R. (1997) Nat. Med. 3, 320–323[CrossRef][Medline] [Order article via Infotrieve]
  4. Calandra, T., Echtenacher, B., Roy, D. L., Pugin, J., Metz, C. N., Hultner, L., Heumann, D., Mannel, D., Bucala, R., and Glauser, M. P. (2000) Nat. Med. 6, 164–170[CrossRef][Medline] [Order article via Infotrieve]
  5. de Jong, Y. P., Abadia-Molina, A. C., Satoskar, A. R., Clarke, K., Rietdijk, S. T., Faubion, W. A., Mizoguchi, E., Metz, C. N., Alsahli, M., ten Hove, T., Keates, A. C., Lubetsky, J. B., Farrell, R. J., Michetti, P., van Deventer, S. J., Lolis, E., David, J. R., Bhan, A. K., Terhorst, C., and Sahli, M. A. (2001) Nat. Immunol. 2, 1061–1066[CrossRef][Medline] [Order article via Infotrieve]
  6. Onodera, S., Kaneda, K., Mizue, Y., Koyama, Y., Fujinaga, M., and Nishihira, J. (2000) J. Biol. Chem. 275, 444–450[Abstract/Free Full Text]
  7. Lue, H., Kleemann, R., Calandra, T., Roger, T., and Bernhagen, J. (2002) Microbes Infect. 4, 449–460[CrossRef][Medline] [Order article via Infotrieve]
  8. Bacher, M., Metz, C. N., Calandra, T., Mayer, K., Chesney, J., Lohoff, M., Gemsa, D., Donnelly, T., and Bucala, R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7849–7854[Abstract/Free Full Text]
  9. Calandra, T., Bernhagen, J., Metz, C. N., Spiegel, L. A., Bacher, M., Donnelly, T., Cerami, A., and Bucala, R. (1995) Nature. 377, 68–71[CrossRef][Medline] [Order article via Infotrieve]
  10. Rosengren, E., Bucala, R., Aman, P., Jacobsson, L., Odh, G., Metz, C. N., and Rorsman, H. (1996) Mol. Med. 2, 143–149[Medline] [Order article via Infotrieve]
  11. Kleemann, R., Kapurniotu, A., Frank, R. W., Gessner, A., Mischke, R., Flieger, O., Juttner, S., Brunner, H., and Bernhagen, J. (1998) J. Mol. Biol. 280, 85–102[CrossRef][Medline] [Order article via Infotrieve]
  12. Taylor, A. B., Johnson, W. H., Jr., Czerwinski, R. M., Li, H. S., Hackert, M. L., and Whitman, C. P. (1999) Biochemistry 38, 7444–7452[CrossRef][Medline] [Order article via Infotrieve]
  13. Sun, H. W., Bernhagen, J., Bucala, R., and Lolis, E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5191–5196[Abstract/Free Full Text]
  14. Suzuki, M., Sugimoto, H., Nakagawa, A., Tanaka, I., Nishihira, J., and Sakai, M. (1996) Nat. Struct. Biol. 3, 259–266[CrossRef][Medline] [Order article via Infotrieve]
  15. Bendrat, K., Al-Abed, Y., Callaway, D. J., Peng, T., Calandra, T., Metz, C. N., and Bucala, R. (1997) Biochemistry 36, 15356–15362[CrossRef][Medline] [Order article via Infotrieve]
  16. Mischke, R., Kleemann, R., Brunner, H., and Bernhagen, J. (1998) FEBS Lett. 427, 85–90[CrossRef][Medline] [Order article via Infotrieve]
  17. Villadangos, J. A., Bryant, R. A., Deussing, J., Driessen, C., Lennon-Dumenil, A. M., Riese, R. J., Roth, W., Saftig, P., Shi, G. P., Chapman, H. A., Peters, C., and Ploegh, H. L. (1999) Immunol. Rev. 172, 109–120[CrossRef][Medline] [Order article via Infotrieve]
  18. Collins, D. S., Unanue, E. R., and Harding, C. V. (1991) J. Immunol. 147, 4054–4059[Abstract]
  19. Haque, M. A., Hawes, J. W., and Blum, J. S. (2001) J. Immunol. 166, 4543–4551[Abstract/Free Full Text]
  20. Haque, M. A., Li, P., Jackson, S. K., Zarour, H. M., Hawes, J. W., Phan, U. T., Maric, M., Cresswell, P., and Blum, J. S. (2002) J. Exp. Med. 195, 1267–1277[Abstract/Free Full Text]
  21. Jensen, P. E. (1991) J. Exp. Med. 174, 1121–1130[Abstract/Free Full Text]
  22. Maric, M., Arunachalam, B., Phan, U. T., Dong, C., Garrett, W. S., Cannon, K. S., Alfonso, C., Karlsson, L., Flavell, R. A., and Cresswell, P. (2001) Science 294, 1361–1365[Abstract/Free Full Text]
  23. Santambrogio, L., Sato, A. K., Carven, G. J., Belyanskaya, S. L., Strominger, J. L., and Stern, L. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15056–15061[Abstract/Free Full Text]
  24. Monji, T., and Pious, D. (1997) J. Immunol. 158, 3155–3164[Abstract]
  25. Fox, B. S., Carbone, F. R., Germain, R. N., Paterson, Y., and Schwartz, R. H. (1988) Nature 331, 538–540[CrossRef][Medline] [Order article via Infotrieve]
  26. Accapezzato, D., Nisini, R., Paroli, M., Bruno, G., Bonino, F., Houghton, M., and Barnaba, V. (1998) J. Immunol. 160, 5262–5266[Abstract/Free Full Text]
  27. Vacchino, J. F., and McConnell, H. M. (2001) J. Immunol. 166, 6680–6685[Abstract/Free Full Text]
  28. Suzuki, M., Murata, E., Tanaka, I., Nishihira, J., and Sakai, M. (1994) J. Mol. Biol. 235, 1141–1143[CrossRef][Medline] [Order article via Infotrieve]
  29. Mischke, R., Gessner, A., Kapurniotu, A., Juttner, S., Kleemann, R., Brunner, H., and Bernhagen, J. (1997) FEBS Lett. 414, 226–232[CrossRef][Medline] [Order article via Infotrieve]
  30. Chicz, R. M., Urban, R. G., Lane, W. S., Gorga, J. C., Stern, L. J., Vignali, D. A., and Strominger, J. L. (1992) Nature. 358, 764–768[CrossRef][Medline] [Order article via Infotrieve]
  31. Chicz, R. M., Graziano, D. F., Trucco, M., Strominger, J. L., and Gorga, J. C. (1997) J. Immunol. 159, 4935–4942[Abstract]
  32. Lee, K. H., Wucherpfennig, K. W., and Wiley, D. C. (2001) Nat. Immunol. 2, 501–507[CrossRef][Medline] [Order article via Infotrieve]
  33. O'Sullivan, D., Sidney, J., Appella, E., Walker, L., Phillips, L., Colon, S. M., Miles, C., Chesnut, R. W., and Sette, A. (1990) J. Immunol. 145, 1799–1808[Abstract]
  34. Gorga, J. C., Horejsi, V., Johnson, D. R., Raghupathy, R., and Strominger, J. L. (1987) J. Biol. Chem. 262, 16087–16094[Abstract/Free Full Text]
  35. Malik, P., and Strominger, J. L. (2000) J. Immunol. Methods 234, 83–88[CrossRef][Medline] [Order article via Infotrieve]
  36. Brocke, P., Garbi, N., Momburg, F., and Hammerling, G. J. (2002) Curr. Opin. Immunol. 14, 22–29[CrossRef][Medline] [Order article via Infotrieve]
  37. Engering, A., and Pieters, J. (2001) Int. Immunol. 13, 127–134[Abstract/Free Full Text]
  38. Kropshofer, H., Spindeldreher, S., Rohn, T. A., Platania, N., Grygar, C., Daniel, N., Wolpl, A., Langen, H., Horejsi, V., and Vogt, A. B. (2002) Nat. Immunol. 3, 61–68[CrossRef][Medline] [Order article via Infotrieve]
  39. Kang, S. J., and Cresswell, P. (2002) EMBO J. 21, 1650–1660[CrossRef][Medline] [Order article via Infotrieve]
  40. Stern, L. J., Brown, J. H., Jardetzky, T. S., Gorga, J. C., Urban, R. G., Strominger, J. L., and Wiley, D. C. (1994) Nature 368, 215–221[CrossRef][Medline] [Order article via Infotrieve]
  41. Auger, I., Escola, J. M., Gorvel, J. P., and Roudier, J. (1996) Nat. Med. 2, 306–310[CrossRef][Medline] [Order article via Infotrieve]
  42. Potolicchio, I., Festucci, A., Hausler, P., and Sorrentino, R. (1999) Eur. J. Immunol. 29, 2140–2147[CrossRef][Medline] [Order article via Infotrieve]
  43. Lubetsky, J. B., Swope, M., Dealwis, C., Blake, P., and Lolis, E. (1999) Biochemistry 38, 7346–7354[CrossRef][Medline] [Order article via Infotrieve]
  44. Dios, A., Mitchell, R. A., Aljabari, B., Lubetsky, J., O'Connor, K., Liao, H., Senter, P. D., Manogue, K. R., Lolis, E., Metz, C., Bucala, R., Callaway, D. J., and Al-Abed, Y. (2002) J. Med. Chem. 45, 2410–2416[CrossRef][Medline] [Order article via Infotrieve]
  45. Zhang, X., and Bucala, R. (1999) Bioorg. Med. Chem. Lett. 9, 3193–3198[CrossRef][Medline] [Order article via Infotrieve]
  46. Phan, U. T., Lackman, R. L., and Cresswell, P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 12298–12303[Abstract/Free Full Text]
  47. Arunachalam, B., Pan, M., and Cresswell, P. (1998) J. Immunol. 160, 5797–5806[Abstract/Free Full Text]
  48. Fiebiger, E., Maehr, R., Villadangos, J., Weber, E., Erickson, A., Bikoff, E., Ploegh, H. L., and Lennon-Dumenil, A. M. (2002) J. Exp. Med. 196, 1263–1270[Abstract/Free Full Text]
  49. Parks, W. C. (2002) J. Clin. Invest. 110, 613–614[CrossRef][Medline] [Order article via Infotrieve]
  50. McCawley, L. J., and Matrisian, L. M. (2001) Curr. Opin. Cell Biol. 13, 534–540[CrossRef][Medline] [Order article via Infotrieve]
  51. Ansorge, S., Schon, E., and Kunz, D. (1991) Biomed. Biochim. Acta 50, 799–807[Medline] [Order article via Infotrieve]
  52. Angelini, G., Gardella, S., Ardy, M., Ciriolo, M. R., Filomeni, G., Di Trapani, G., Clarke, F., Sitia, R., and Rubartelli, A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 1491–1496[Abstract/Free Full Text]
  53. Srivastava, P. K., Menoret, A., Basu, S., Binder, R. J., and McQuade, K. L. (1998) Immunity 8, 657–665[CrossRef][Medline] [Order article via Infotrieve]
  54. Wang, X. Y., Kazim, L., Repasky, E. A., and Subjeck, J. R. (2001) J. Immunol. 166, 490–497[Abstract/Free Full Text]
  55. Li, Z., Menoret, A., and Srivastava, P. (2002) Curr. Opin. Immunol. 14, 45–51[CrossRef][Medline] [Order article via Infotrieve]
  56. Auger, I., Lepecuchel, L., and Roudier, J. (2002) Arthritis Rheum. 46, 929–933[CrossRef][Medline] [Order article via Infotrieve]
  57. Potolicchio, I., Brookes, P. A., Madrigal, A., Lechler, R. I., and Sorrentino, R. (1996) Transplantation 62, 1347–1352[CrossRef][Medline] [Order article via Infotrieve]
  58. Richeldi, L., Sorrentino, R., and Saltini, C. (1993) Science 262, 242–244[Abstract/Free Full Text]
  59. Potolicchio, I., Mosconi, G., Forni, A., Nemery, B., Seghizzi, P., and Sorrentino, R. (1997) Eur. J. Immunol. 27, 2741–2743[Medline] [Order article via Infotrieve]
  60. Santambrogio, L., Sato, A. K., Fischer, F. R., Dorf, M. E., and Stern, L. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15050–15055[Abstract/Free Full Text]
  61. Chervonsky, A. V., Medzhitov, R. M., Denzin, L. K., Barlow, A. K., Rudensky, A. Y., and Janeway, C. A., Jr. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10094–10099[Abstract/Free Full Text]
  62. Mullen, M. M., Haan, K. M., Longnecker, R., and Jardetzky, T. S. (2002) Mol. Cell 9, 375–385[CrossRef][Medline] [Order article via Infotrieve]
  63. Basu, S., Binder, R. J., Ramalingam, T., and Srivastava, P. K. (2001) Immunity 14, 303–313[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
M. Winner, A. C. Koong, B. E. Rendon, W. Zundel, and R. A. Mitchell
Amplification of Tumor Hypoxic Responses by Macrophage Migration Inhibitory Factor-Dependent Hypoxia-Inducible Factor Stabilization
Cancer Res., January 1, 2007; 67(1): 186 - 193.
[Abstract] [Full Text] [PDF]

Home page
 Diabetes CareHome page
C. Herder, H. Kolb, W. Koenig, B. Haastert, S. Muller-Scholze, W. Rathmann, R. Holle, B. Thorand, and H.-E. Wichmann
Association of Systemic Concentrations of Macrophage Migration Inhibitory Factor With Impaired Glucose Tolerance and Type 2 Diabetes: Results from the Cooperative Health Research in the Region of Augsburg, Survey 4 (KORA S4)
Diabetes Care, February 1, 2006; 29(2): 368 - 371.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. T. Nguyen, J. Beck, H. Lue, H. Funfzig, R. Kleemann, P. Koolwijk, A. Kapurniotu, and J. Bernhagen
A 16-Residue Peptide Fragment of Macrophage Migration Inhibitory Factor, MIF-(50-65), Exhibits Redox Activity and Has MIF-like Biological Functions
J. Biol. Chem., September 5, 2003; 278(36): 33654 - 33671.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/33/30889    most recent
M302854200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Potolicchio, I.
Right arrow Articles by Strominger, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Potolicchio, I.
Right arrow Articles by Strominger, J. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement