236- Analysis of mouse mannan-binding lectin A aad C Hong Liu, L~sbeth Jensen, Smen Hansen, Steen Vang P&mm, Kazue Takahashi, Alan Ezekowtz, Fred&k Hams Jens C. Jensaim Steffen Thxl (st:rj:microbiolo~~.~u.~) Deparmtent of Medical Mcrobiologv and Immunology, Universily ofAarhus, DK-Denmark

Mannan-binding leetin (MBL) is a serum protein derived from liver and it belongs to the family of Ca2’-dependent collagenous lectin. Only one form of human MBL has been characterized, while two forms of MBL have been found in rabbits. rats, mice and rhesus monkeys. Rat monoclonal antibodies towards MBL-A and -C are generated. All the antibodies react with MBLs at both reduced and non-reduced conditions. Two time resolved wnmunojlourometnc assays (TRIFMA), lectin- and antigen- TRIFMA, have been constructed. The assays have a detection level below 100 pg/ml with a working range of 3 orders of magnitude. The concentrations of MBL-A and -C are determined at 7 5 and 45 pg/ml, respectively, in a pool of mouse= serum. On gel permeation chromatography, serum MBL-A eluted corresponding to an Mr of 850 Kda, while the majority of MBL-C showed an Mr of 950 Kda. On sucrose gradient centrifugation, the sedimentation rates of MBL-A and - C were determined at 7.3 S and 10.8 S, respectively. The MBL- A and -C levels in ten laboratory mice strains were found to vary between 4 to 12 @ml, and 16 to 118 &ml, respectively. The concentrations of MBLs were followed after induction of acute phase response by intmperitoneal injection of either casein or lipopolysaccharide.

. . . . . . . . . . . . . ...” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -----_--- --- 237- A FICOLIN-LIKE LECTIN IN THE PLASMA OF THE SOLITARY ASCIDIAN, HALOCYN- THZA RORETZZ A. Kenjo, M. Takahashi, M. Matsushita, Y. Endo, T. Fujita. Department of Biochemistry, Fukushima Medical University School of Medicine, Fukushima, Japan.

Ficolins are &tins with collagen-and fibrinogen-like domains and play a role in the first line of host defense against pathogens. To elucidate the origin and evolution of ficolins, the plasma from the solitary ascidian, Halocynfhia roretzi, was applied to affinity chromatography column bearing N-acetylglucosamine (GlcNAc) and eluted with GlcNAc. SDS-PAGE of the eluted proteins revealed a 50 kDa band under reducing conditions. Degenerate primers were designed based on the N-terminal sequence of the protein (PSO) and that of a 30 kDa fragment digested by sraphylococcur aureu V8 protease and used to amplify a part of P50 cDNA born sscidisn hepatopancreas cDNA. To obtain full length of cDNA, 5’ and 3’RACE were conducted. P50 contained a 1,068 bp open reading tie encoding 335 amino acids preceded by a leader peptide of 21 amino acids. Sequence analysis revealed that P50 contained a collagenous region, consisting of Gly-X-Y triplet repeats and that the C-terminal half contained a tibrinogen-like domain. However, collagenous region of PSO was very short (5 repeats) compared to that of human licolinIP35 (15 repeats). The predicted molecular mass of P50 was 38,229 Da, indicating that the mature protein of P50 may be glycosylated. P50 showed 50.8, 49.7, and 47.2 % homology to human ficolinIP35, P35-related protein, and Hakata antigen, respectively, at the amino acid level. These results indicated that P50 is homologous to mammalian ficolins. The ability of the ascidiao ticolin to activate the Fomplement system is currently under investigation.

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236- COMPARISON OF BINDING AND FUNCTIONAL EFFECTS OF MBL AND Clq ON LYMPHOCYTES AND MACROPHAGES

A.Erdei*~.Bajtay*,M.J6~i*Z.Bbnkir, S.TbieW, N.Thielensi *Dept. bnmunol., E&v&L. Univ., G&i, Hungary v Dept. Med. Microbial. and Jmmunol., Aarhus Univ., Denmark &Institute of Structural Biology, Grenoble, France

While the interaction of complement component Clq with cellular proteins is exteosively stwdipd, much lew is known &cut the binding of the structurally related molecule, mannan-binding Win (MBL) to various cells. Here. we demonstrate by cytofluorimetIy that the interaction of MBL with intact immunocompetent cells is much more restricted than that of Clq. It is shown that under conditions of physiological ionic strength MBL binds to human mmocyte&rived M+ and monocytoid cell lines, but not to T- and B-lymphocytes, in contrast to Ciq, which interacts with all these cells under the same amditions. In contrast to the binding of Clq, low ionic strength does not improve the interaction of MBL with MI& No competition for cellular binding sites was found w&en MBL and Clq we added simultane~lsly to the cells. Studying the timctional consequences of the heraction, we found that the release of TNF-a t?om MI$ is induced by Clq but not by MBL. Production of C3 by MO is stimulated by Clq strongly, while the e&ct of MBL is much weaker. C3 produced upon Clq-mediated triggering is shown to opsonize RBC, resulting in enhanced phagocytmis.

These results suggest that cell membrane molecules binding MBL and Clq are not identical moreover, biological functions exerted by these proteins are also markedly different.

239. CONSTRUCTION AND EXPRESSION OF VARI- ANT FORMS OF HUMAN MANNOSE-BINDING LE- CTIN Flemming Larsen’, Hans 0. Madsen’, Claus Koch’ and Peter Garred’, ‘Dept. of Clin. Immunol, Rigshospi- talet, and ‘State Serum Institute. Copenhagen, Denmark.

The normal mannose-binding lectin (MBL) gene (MBL-WT) was cloned by RT-PCR. The naturally occuring variant 52mAfl (MEL-D) was constructed by site-directed mutagenesis. The constructs were inserted into the dicistronic expression vector pEDdC that contains the dihydrofolate reductase gene and co- transfected with pSV,NEO (NEOR) into CHO-DG-44 cells. After selection with G418, the cells were treated with methotrexate to select for high-expression clones. Expression levels similar to human serum levels were obtained.

As judged by western blot, MBL-WT expressed in CHO cells forms higher oligomers of a MW, of approximately 400-600 kDa corresponding to 12-18 subunits, whereas MBL-D forms oligomers of a MW, of 100 kDa corresponding to 3 subunits. MBL-Wl was able to bind mannan and Candida albicans in a Ca” dependent manner, while MBL-D was not. Binding of MBL-WT to mannan could be inhibited by adding mannose or N-acetylglycosamine. When bound to mannan, MBL-WT was able to activate complement judged by deposition of C4 whereas MBL-D could not.

In conclus/on we have constructed and expressed recombinant variants of human MBL. Secretion of MBL-D is similar to that of MBL-WT. but MBL-D is unable to polymerize to higher order oligomers, which probably explain the association of this variant with MBL-related immunodeficiency.