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Laboratoires
*
d’Enzymologie Moléculaire,
Spectrométrie de Masse des Protéines, and
Cristallographie et Cristallogénèse des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel (Commissariat à l’Energie Atomique-Centre National de la Recherche Scientifique-Université Joseph Fourier), Grenoble, France
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Human MBL is assembled from a single polypeptide chain, consisting of a short N-terminal cysteine-rich region, a collagenous region comprising 19 repeating Gly-X-Y triplets with an interruption at the 8 triplet, a 34-residue hydrophobic stretch, and a C-terminal C-type lectin domain. Three polypeptide chains associate to form a homotrimeric structural unit comprising a collagen-like triple helix, an 
-helical coiled-coil called the neck region, and three carbohydrate recognition domains (5). The three cysteines in the N-terminal region form interchain disulfide bonds that mediate formation of oligomers comprising two to six structural units and exhibiting bouquet-like structures when viewed by electron microscopy (14). The collagen-like triple helices associate laterally at their N-terminal ends and then diverge at the level of the interruption in the Gly-X-Y sequence, thus defining a hinge in the molecule.
There are large variations in the MBL serum concentration, arising from polymorphism in the promoter region as well as in the structural moiety of the MBL gene (15), and several clinical studies have provided evidence for a link between a low MBL titer and increased susceptibility to various infectious diseases (16). The structural mutations located in exon 1 of the MBL gene generate amino acid substitutions in the collagen-like region that are believed to impair oligomerization of the protein and result in functional deficiency (15, 17, 18, 19). The reduced ability of these mutants to activate complement has been proposed to result from failure to bind to the MBL-associated serine proteases (20), and from the reduced carbohydrate-binding ability of the lower order MBL oligomers (17, 18, 21).
Purified serum MBL appears to be a mixture of oligomers of different sizes with two major forms, termed MBL-I and MBL-II (8), that can be separated using anion-exchange chromatography (8, 22). The objective of the present study was to precisely characterize the oligomerization state of both forms, their carbohydrate-binding ability, and their interaction properties with the MASPs and MAp19. Mass spectrometry analysis provides unambiguous evidence that MBL-I and -II are trimers and tetramers of the structural unit, respectively. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, with the tetrameric form showing increased binding capacity and higher affinity, but has no significant influence on the interaction with the MASPs and MAp19 or on the stoichiometry of the resulting complexes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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N-Acetylglucosamine-BSA (10 average carbohydrate residues per BSA molecule) was purchased from Dextra Laboratories. Mannose (Man)- and galactose-BSA (30 average carbohydrate residues per BSA molecule) were from EY Laboratories.
Proteins
MBL was purified from human serum as described by Thielens et al. (23). Separation of MBL-I and MBL-II was performed by anion-exchange chromatography on a MonoQ HR 5/5 column (Amersham Pharmacia Biotech) as described by Dahl et al. (8) with the following modifications. The column was run at a flow rate of 0.5 ml/min, and MBL fractionation was achieved with a NaCl gradient to 250 mM in 40 min. Fractions of 1 ml were collected and analyzed for the presence of MBL-I and MBL-II by SDS-PAGE and Coomassie Blue staining. Recombinant MASP-1, MASP-2, and MAp19 were expressed using a baculovirus/insect cell system and purified as described previously (23, 24). Recombinant MASP-3 was expressed using the same system and purified as described by Zundel et al. (25) with the following changes. After the first anion-exchange chromatography step, fractions containing MASP-3 were dialyzed against 50 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine-hydrochloride, pH 6.9, and loaded at 1.0 ml/min onto a MonoQ HR 5/5 column (Amersham Biosciences) equilibrated in the same buffer. Elution was conducted with a linear NaCl gradient to 500 mM in 1 h. MASP-3-containing fractions were identified by SDS-PAGE analysis; they were dialyzed against 145 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 7.4; and further purification was achieved by high pressure gel permeation on a TSK G3000 SWG column (7.5 x 600 nm) (Tosohaas) equilibrated in the same buffer.
The concentrations of purified MBL-I, MBL-II, MASP-3, and MAp19 were determined using molar extinction coefficients at 280 nm of 198,000, 264,000, 160,600, and 29,000 M–1 x cm–1, respectively, which were determined experimentally by amino acid analysis. The molecular masses of MBL-I (228,098 Da), MBL-II (304,899 Da), MASP-3 (87,524 Da), and MAp19 (19,088 Da) were determined by MALDI-TOF mass spectrometry analysis. Due to the low amount of material recovered, estimation of the concentration of recombinant MASP-1 and MASP-2 was based on Coomassie Blue staining after SDS-PAGE analysis using MASP-3 and MAp19 as internal standards and molecular masses of 82,000 and 75,100 Da, respectively (24).
Amino acid and N-terminal sequence analyses
Amino acid analyses were performed on acid hydrolysates of the samples using a Beckman 7300 amino acid analyzer as described previously (26). N-terminal sequence analyses were performed using an Applied Biosystems model 477A protein sequencer as described previously (27).
MALDI-TOF mass spectrometry analysis
Mass spectra were obtained using a Perseptive Biosystems Voyager Elite Xl time of flight mass spectrometer with delayed extraction, operating with a pulsed nitrogen laser at 337 nm. Positive ion mass spectra were acquired using linear, delayed extraction mode with an accelerating potential of 25 kV, an 85% grid potential, a 0.3% guide wire voltage, and a delay time of 600 ns. Samples were mixed with an equal volume of a saturated solution of sinapinic acid prepared in 50% (v/v) acetonitrile, 0.1% trifluoroacetic acid on the stainless steel sample plate and air-dried before analysis. External calibration was performed with BSA using m/z values of 66,431 and 33,216 for the mono- and doubly-charged species, respectively. The accuracy of molecular mass determination was estimated at 0.075%.
Gel permeation chromatography
Complexes between trimeric or tetrameric MBL on one hand and MASP-3 or MAp19 on the other were assembled by incubation of the proteins for 1 h at room temperature and analyzed by gel permeation chromatography on a Superose 6 10/300 GL column (Amersham Biosciences) equilibrated in 145 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 7.4, and run at 0.5 ml/min. Proteins were detected from their optical density at 280 nm.
Real time surface plasmon resonance spectroscopy and data evaluation
Analyses were performed using a Biacore 3000 instrument (Biacore AB). Carbohydrate-BSA glycoconjugates and MBL were diluted to 10 and 30 µg/ml, respectively, in 10 mM sodium acetate, pH 4.0, and immobilized on the surface of a CM5 sensor chip (Biacore AB) using amine-coupling chemistry as described by Zundel et al. (25). Binding of trimeric and tetrameric MBL to immobilized Man-BSA (2400 resonance units (RU)), GlcNAc-BSA (3000 RU), or Gal-BSA (2300 RU) was measured at a flow rate of 20 µl/min in 145 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 7.4, containing 0.005% surfactant P20 (Biacore AB). Binding of MASP-1, MASP-2, MASP-3, and MAp19 to immobilized trimeric MBL (9,000 RU) and tetrameric MBL (10,000 RU) was measured under the same conditions. Equivalent volumes of each protein sample were injected over a surface with immobilized BSA to serve as blank sensorgrams for subtraction of the bulk refractive index background. Regeneration of the surfaces was achieved by injection of 10 µl of the running buffer containing 5 mM EDTA instead of 1 mM CaCl2.
Data were analyzed by global fitting to a 1:1 Langmuir binding model of both the association and dissociation phases for several concentrations simultaneously, using the BIAevaluation 3.1 software (Biacore AB). The apparent equilibrium dissociation constants (KD) were calculated from the ratio of the dissociation and association rate constants (koff/kon). Maximal binding capacities (Rmax) were determined using the same model. The 
2 value, which is a standard statistical measure of the closeness of the fit (BIAevaluation 3 Software Handbook), was <2 in all cases.
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Results |
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Purified human serum MBL appears as a mixture of oligomers of different sizes that can be fractionated using anion-exchange chromatography (8). SDS-PAGE analysis of the two major MBL species isolated from serum, MBL-I and MBL-II, showed that they migrate as diffuse bands with apparent molecular masses of 
235 and 310 kDa, respectively, under nonreducing conditions (Fig. 1A). In the case of MBL-II (Fig. 1, lane 2), two additional minor bands were detected, one corresponding to trace amounts of MBL-I and the other one with an apparent molecular mass of 385 kDa, corresponding to the higher order oligomeric species termed MBL-III by Dahl et al. (8). All forms yielded a single chain with an apparent molecular mass of 32 kDa under reducing conditions (Fig. 1B).
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The carbohydrate-binding ability of trimeric and tetrameric MBL was investigated by surface plasmon resonance spectroscopy, using glycoconjugates (Man-BSA and GlcNAc-BSA) as the immobilized ligands. As shown in Fig. 4, each MBL form bound to Man and N-acetylglucosamine in the presence of 1 mM CaCl2. Complete elution of the bound protein could be achieved in all cases by a pulse injection of EDTA or of a 0.3 M solution of mannose or GlcNAc. The binding curves exhibited quite different shapes, with a higher binding level and a slower dissociation of tetrameric MBL for both glycoconjugates (Fig. 4). The kinetic parameters of the interactions were determined by recording sensorgrams at varying MBL concentrations and evaluation of the data by global fitting as described in Materials and Methods. The values of maximal binding capacity (Rmax), association (kon) and dissociation (koff) rate constants and the resulting apparent KD values are listed in Table III. Consistent with our preliminary analyses (see Fig. 4), tetrameric MBL exhibited much lower koff values than trimeric MBL for binding to both Man and GlcNAc (13- and 3-fold, respectively). In contrast, trimeric MBL exhibited slightly higher kon values than tetrameric MBL for binding to either carbohydrate. The resulting apparent KD values were in the nanomolar range in all instances, with comparable values for both MBL forms in the case of GlcNAc and a 4-fold higher affinity for tetrameric MBL in the case of mannose (Table III). Tetrameric MBL showed a 4- and 12-fold higher maximal binding capacity (Rmax) for Man and GlcNAc, respectively, compared with the trimeric form (Table III).
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Interaction properties of trimeric and tetrameric MBL with the MASPs and MAp19
Trimeric and tetrameric MBL were next immobilized on a sensorchip to compare their interaction properties with the recombinant MASPs and MAp19 used as soluble ligands. As shown in Fig. 5, MASP-1 (Fig. 5A), MASP-2 (Fig. 5B), MASP-3 (Fig. 5C), and MAp19 (Fig. 5D) each individually bound to immobilized trimeric or tetrameric MBL in very similar ways, with virtually identical association and dissociation phases in both cases. Likewise, as previously determined in the case of MASP-3 (25), the kinetic rate constants for MASP-1, MASP-2, and MAp19 were of the same order in the case of trimeric and tetrameric MBL, and the resulting KD values exhibited no significant difference (Table IV). No binding of the ligands to either trimeric or tetrameric MBL was observed when EDTA was substituted for CaCl2 in the running buffer.
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To further characterize the interaction properties of trimeric and tetrameric MBL, individual MBL-MASP complexes were assembled from the isolated components, and their molecular composition was studied using gel permeation chromatography. Due to the low amount of recombinant MASP-1 and MASP-2 available, experiments were performed using MASP-3 and MAp19. Both proteins have been shown to form dimers in the presence of Ca2+ ions (23, 25). Separate analyses of trimeric MBL and the MASP-3 dimer were first performed, indicating that the isolated proteins eluted at 24.1 and 25.2 min, respectively (Fig. 6A). When trimeric MBL and MASP-3 were mixed in a 1:2 molar ratio and then applied to the column, no residual peak corresponding to either protein was detected, but a new peak eluting ahead of trimeric MBL was observed, consistent with the formation of a 1:2 MBL/MASP-3 complex (Fig. 6A). When trimeric MBL and MASP-3 were mixed in a 1:1 or 1:4 ratio, the amount of complex formed and its elution position did not change, whereas extra peaks corresponding to either excess trimeric MBL or excess MASP-3 were observed. A minor peak eluting at 31 min and showing no interaction with MASP-3 was also detected in the trimeric MBL sample (Fig. 6A). This peak was unlikely to correspond to a MBL form of lower mass (i.e., with only one or two structural units) because no such species was detected to a significant extent by SDS-PAGE analysis of MBL-I (Fig. 1A). Analysis of the interaction between MASP-3 and tetrameric MBL yielded results very similar to those obtained with trimeric MBL, fully consistent with the formation of a 1:2 MBL/MASP-3 complex, even in the presence of a molar excess of MASP-3 (Fig. 6B).
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Discussion |
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The mass value determined for the reduced MBL chain by MALDI-TOF mass spectrometry is close to the 25,500-Da value obtained by Larsen et al. (21), using surface-enhanced laser desorption/ionization mass spectrometry. Compared with the unmodified polypeptide, the MBL chain shows a mass increase that accounts for the posttranslational modifications usually observed in collagens, i.e., hydroxylation of the proline and lysine residues in the Y position of the repeating Gly-X-Y sequence and glycosylation of hydroxylysine residues. Previous analyses have indicated the presence of 3.7–4 hydroxyproline and 3.2–4 hydroxylysine residues per MBL chain (31, 32). Our own results are consistent with the presence of 4 hydroxyproline and 4 hydroxylysine residues, among which 3–4 carry the characteristic O-linked glucosylgalactosyl disaccharide observed in other proteins containing collagen-like sequences such as rat MBL (33), human C1q (34), or adiponectin (35). The fact that the mass values for the elementary chain of trimeric and tetrameric MBL are strictly identical indicates that preferential association into trimers or tetramers is not dependent on the extent of modification in the collagen-like region.
Analysis of the carbohydrate-binding properties of trimeric and tetrameric MBL reveals that they bind to Man-BSA with KD values of 2.2 and 0.55 nM and to GlcNAc-BSA with KD values of 1.2 and 0.96 nM, respectively, indicating high affinity in all cases. These values are in the same range as those determined previously for binding of rat MBL to glycosylated BSA (1 nM) (36) or of human MBL to mannan (2.3 nM) (37). Although the apparent KD values are of the same order for trimeric and tetrameric MBL, these two forms show strikingly different kinetic rate constants. Thus, tetrameric MBL exhibits a 13-fold lower dissociation rate constant from Man-BSA than trimeric MBL does, reflecting a much higher stability of the carbohydrate-tetrameric MBL interaction. Although the kon value is 3-fold lower in the case of tetrameric MBL, the maximal binding capacity to Man-BSA is 4-fold higher, and therefore much more mannose/tetrameric MBL complexes are expected to be formed in the presence of equivalent concentrations of both MBL forms (Fig. 4A). Comparable results were obtained using GlcNAc-BSA as a ligand, although in that case the major difference is that tetrameric MBL has a 12-fold higher maximal binding capacity than trimeric MBL. Thus, the oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, with the tetrameric form showing increased binding capacity and higher interaction stability. It was shown previously that monovalent interaction between a single carbohydrate recognition domain of rat MBL-A and mannose is characterized by a very weak affinity (KD in the millimolar range) (38). Interaction of oligomeric MBL with carbohydrate arrays allows simultaneous engagement of several lectin domains, hence strengthening binding through an avidity phenomenon. In this respect, our data clearly indicate that the tetravalent MBL form has more avidity than the trivalent MBL form. Consistent with these observations, it was reported recently that mutated MBL molecules with a dimeric structure bind mannan with a markedly decreased capacity compared with wild-type MBL (21).
MBL-I and MBL-II were found to be preferentially associated in serum with MASP-1 and MAp19 and with MASP-3 and MASP-2, respectively (8). In contrast, using surface plasmon resonance spectroscopy, we have shown that recombinant MASP-3 binds to serum trimeric and tetrameric MBL with comparable binding constants (25). We have now extended this study to recombinant MASP-1, MASP-2, and MAp19, and we show that these proteins each interact individually with trimeric and tetrameric MBL with similar binding constants. Our data therefore indicate that the oligomerization state of MBL has no significant effect on its MASP-binding properties in vitro. In this respect, it was reported recently that purified tetrameric MBL induces C4 cleavage more efficiently than does trimeric MBL when added to MBL-deficient plasma (22), and it was suggested that this arises from a preferential association of serum MASP-2 with higher MBL oligomers. In light of our data, it should be pointed out that this difference may also be accounted for by the differential carbohydrate-binding capacity of trimeric and tetrameric MBL, because the complex between trimeric MBL and MASP-2 would exhibit a reduced capacity to bind to the mannan surface used in these experiments and therefore would trigger MASP-2 activation and C4 cleavage less efficiently.
Gel filtration analysis of the complexes between trimeric or tetrameric MBL and MASP-3 or MAp19 reveals a 1:2 stoichiometry in all cases. Although similar experiments could not be done using MASP-1 and MASP-2, it should be stressed that MASP-1 and MASP-3 share the same N-terminal CUB1-epidermal growth factor (EGF)-CUB2 domains known to mediate binding to human MBL or rat MBL-A (24, 39). In agreement with the observation that these proteases bind trimeric and tetrameric MBL with comparable affinities (Table IV), it can be inferred that MASP-1 associates with either MBL species with the same stoichiometry as determined for MASP-3. This reasoning likely also applies to MASP-2, because this protease comprises the same N-terminal CUB1-EGF modules as MAp19 (12, 13). The present results do not fully agree with those of Chen and Wallis (40) who reported that, whereas rat recombinant MBL-A dimers bind two MASP-1 or MASP-2 CUB1-EGF-CUB2 fragments, trimeric and tetrameric MBL-A forms bind up to four. It should be emphasized, however, that the latter complexes were observed only in the presence of a large molar excess (6- to 12-fold) of the MASP fragments over MBL-A.
The x-ray structure of human MAp19 has been solved recently, and site-directed mutagenesis studies have allowed us to map the MBL-binding site at the distal end of the CUB1 module (41). A three-dimensional model of the interaction between the MAp19 dimer and a triple-helical segment of MBL containing the putative MASP-binding site was proposed. This information was used to construct models of the trimeric and tetrameric MBL molecules and of their complexes with MAp19. As illustrated in Fig. 8B, the complex between the MBL tetramer and the MAp19 dimer is expected to be symmetrical, the latter protein interacting with binding sites located on two opposite collagen triple helices of tetrameric MBL. In contrast, it is clear that such a symmetry cannot be conserved in the complex between trimeric MBL and MAp19, because in this case MAp19 should interact with two contiguous collagen helices (Fig. 8A).
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Disclosures |
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Footnotes |
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1 This work was supported by the Commissariat à l’Energie Atomique, the Centre National de la Recherche Scientifique, and the Université Joseph Fourier (Grenoble, France). A preliminary report of this study was presented by N.M.T. at the 20th International Complement Workshop in Honolulu, HI, July 13–18, 2004. 
2 Address correspondence and reprint requests to Dr. Nicole Thielens, Laboratoire d’Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France. E-mail address: nicole.thielens{at}ibs.fr
3 Abbreviations used in this paper: MBL, mannan-binding lectin; MASP, MBL-associated serine protease; GlcNAc, N-acetylglucosamine; MAp19, 19-kDa MBL-associated protein; CUB module, module originally found in complement proteins C1r/C1s, Uegf, and bone morphogenetic protein-1; EGF, epidermal growth factor; Man, mannose; RU, resonance unit(s); the nomenclature of protein modules is that defined by Bork and Bairoch (1 ).
Received for publication October 6, 2004. Accepted for publication December 6, 2004.
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References |
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Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease. Biochem. J. 311:1021.
fragments of human complement proteases C1-r and C1-s. J. Biol. Chem. 265:14469.-helical coiled-coil. Nat. Struct. Biol. 1:789.[Medline]This article has been cited by other articles:
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  A. E. Phillips, J. Toth, A. W. Dodds, U. V. Girija, C. M. Furze, E. Pala, R. B. Sim, K. B. M. Reid, W. J. Schwaeble, R. Schmid, et al. Analogous Interactions in Initiating Complexes of the Classical and Lectin Pathways of Complement J. Immunol., June 15, 2009; 182(12): 7708 - 7717. [Abstract] [Full Text] [PDF]
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M. Lacroix, C. Dumestre-Perard, G. Schoehn, G. Houen, J.-Y. Cesbron, G. J. Arlaud, and N. M. Thielens Residue Lys57 in the Collagen-Like Region of Human L-Ficolin and Its Counterpart Lys47 in H-Ficolin Play a Key Role in the Interaction with the Mannan-Binding Lectin-Associated Serine Proteases and the Collectin Receptor Calreticulin J. Immunol., January 1, 2009; 182(1): 456 - 465. [Abstract] [Full Text] [PDF]
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 F. Teillet, C. Gaboriaud, M. Lacroix, L. Martin, G. J. Arlaud, and N. M. Thielens Crystal Structure of the CUB1-EGF-CUB2 Domain of Human MASP-1/3 and Identification of Its Interaction Sites with Mannan-binding Lectin and Ficolins J. Biol. Chem., September 12, 2008; 283(37): 25715 - 25724. [Abstract] [Full Text] [PDF]
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N. Rawal, R. Rajagopalan, and V. P. Salvi Activation of Complement Component C5: COMPARISON OF C5 CONVERTASES OF THE LECTIN PATHWAY AND THE CLASSICAL PATHWAY OF COMPLEMENT J. Biol. Chem., March 21, 2008; 283(12): 7853 - 7863. [Abstract] [Full Text] [PDF]
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V. Garlatti, L. Martin, E. Gout, J.-B. Reiser, T. Fujita, G. J. Arlaud, N. M. Thielens, and C. Gaboriaud Structural Basis for Innate Immune Sensing by M-ficolin and Its Control by a pH-dependent Conformational Switch J. Biol. Chem., December 7, 2007; 282(49): 35814 - 35820. [Abstract] [Full Text] [PDF]
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 S. M. Van Patten, H. Hughes, M. R. Huff, P. A. Piepenhagen, J. Waire, H. Qiu, C. Ganesa, D. Reczek, P. V. Ward, J. P. Kutzko, et al. Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease Glycobiology, May 1, 2007; 17(5): 467 - 478. [Abstract] [Full Text] [PDF]
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F. Teillet, M. Lacroix, S. Thiel, D. Weilguny, T. Agger, G. J. Arlaud, and N. M. Thielens Identification of the Site of Human Mannan-Binding Lectin Involved in the Interaction with Its Partner Serine Proteases: The Essential Role of Lys55 J. Immunol., May 1, 2007; 178(9): 5710 - 5716. [Abstract] [Full Text] [PDF]
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M. Dong, S. Xu, C. L. P. Oliveira, J. S. Pedersen, S. Thiel, F. Besenbacher, and T. Vorup-Jensen Conformational Changes in Mannan-Binding Lectin Bound to Ligand Surfaces J. Immunol., March 1, 2007; 178(5): 3016 - 3022. [Abstract] [Full Text] [PDF]
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C. Yamada, H. Sano, T. Shimizu, H. Mitsuzawa, C. Nishitani, T. Himi, and Y. Kuroki Surfactant Protein A Directly Interacts with TLR4 and MD-2 and Regulates Inflammatory Cellular Response: IMPORTANCE OF SUPRATRIMERIC OLIGOMERIZATION J. Biol. Chem., August 4, 2006; 281(31): 21771 - 21780. [Abstract] [Full Text] [PDF]
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V. Rossi, F. Teillet, N. M. Thielens, I. Bally, and G. J. Arlaud Functional Characterization of Complement Proteases C1s/Mannan-binding Lectin-associated Serine Protease-2 (MASP-2) Chimeras Reveals the Higher C4 Recognition Efficacy of the MASP-2 Complement Control Protein Modules J. Biol. Chem., December 23, 2005; 280(51): 41811 - 41818. [Abstract] [Full Text] [PDF]
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R. Wallis, N. J. Lynch, S. Roscher, K. B. Reid, and W. J. Schwaeble Decoupling of Carbohydrate Binding and MASP-2 Autoactivation in Variant Mannose-Binding Lectins Associated with Immunodeficiency J. Immunol., November 15, 2005; 175(10): 6846 - 6851. [Abstract] [Full Text] [PDF]
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