Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-μTP-L309C) generated by fusion of the IgM μ-tailpiece to the C terminus of human IgG1 Fc. Fc-μTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-μTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-μTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-μTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-μTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.

ABSTRACT

Arabinogalactan-proteins (AGPs) are proteoglycans containing a high proportion ofcarbohydrate (typically >90%) linked to a protein backbone rich in hydroxyproline (Hyp), Ala Approximately 93% of the Pro residues are hydroxylated and hence are potential sites for glycosylation.Arabinogalactan-proteins (AGPs) occur predominantly in the intercellular spaces of plant tissues but are also associated with membranes, some cytoplasmic organelles, and the cell wall (for reviews see refs. 1-5). AGPs bind to and are precipitated by the P-glucosyl Yariv reagent (6). The function of AGPs is not established, but they may be involved in development, cell-cell interactions, and plant defense.The carbohydrate component of AGPs is generally composed of arabinose and galactose with minor amounts of other sugars. Linkage analysis is consistent with a structure based on a 3-linked (3-galactosyl backbone, branched through C(0)6 to 6-linked galactosyl side chains. The arabinose is most often present as terminal residues. The protein is usually a minor component with characteristically high levels of hydroxyproline (Hyp), Ala, and Ser (for exceptions see refs. 7 and 8). Relatively little is known about the structure ofthe protein core ofAGPs; only a few peptide sequences are available (7,(9)(10)(11) AGPs were deglycosylated using anhydrous HF (18) and fractionated by size-exclusion FPLC and RP-HPLC according to Fig. 1 D-E. The protein backbones were digested with thermolysin, and the products were separated on a C18 microbore HPLC column (Ultrasphere ODS, 2.1 x 250 mm) and eluted with a gradient of acetonitrile in 0.1% aqueous TFA. Individual peaks were repurified and sequenced (19). Amino acid analyses were performed as described by Simpson et al. (20).Isolation of cDNA Clones. A 68-base oligonucleotide, 5'-GCAAAATCACCAACAGCAACACCACCAACAGCAA-CACCACCATCAGCAGTATATAGTGAGTCGTATTA-3', was synthesized. The first part of the sequence codes for the AGP peptide A-K-S-O-T-A-T-O-O-T-A-T-O-O-S-A-V (

SummaryThis paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGPP¢2) and the other from suspension cultures of Nicotiana alata (AGPNa2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGPP¢2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGPNa2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Sar, Ala, Thr and the other rich in Asn, Tyr and Sar. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the "classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well