Annie Galinha

Identification of the cleavage site involved in production of plasma soluble Fc gamma receptor type III (CD16)

CD16 (Fcγ R type III) is a low‐affinity IgG Fc receptor (R) that exists in two isoforms, a transmembrane FcγRIIIa expressed by NK cells and monocytes, and a phosphatidylinositol‐linked FcγRIIIb expressed by neutrophils. A soluble form of CD16 (sCD16) circulates in plasma. The cleavage site and the nature of the enzyme(s) involved in production of sCD16 were investigated. Soluble CD16 was purified to apparent homogeneity from human serum by eight steps, including anion exchange and immunoaffinity chromatography. Serum sCD16 was sequenced at both ends, as well as a recombinant form of sCD16 used as control. N‐terminal sequencing demonstrated that serum sCD16 originates from neutrophil FcγRIIIb and C‐terminal sequencing suggested that the cleavage site is between Val 196 and Ser 197, close to the membrane anchor. Addition of a hydroxamate‐based inhibitor of Zn2+ metalloproteinases (RU36156) led to a dramatic decrease of sCD16 production by phorbol 12‐myristate 13‐acetate‐activated neutrophils, whereas inhibitors of serine proteinases had no significant effect, showing the metalloproteinase dependence of this cleavage process.

Soluble FcγR (sFcγR) : detection in biological fluids and production of a murine recombinant sFcγR biologically active in vitro and in vivo

Abstract Soluble forms of receptors for the Fc portion of IgG (sFcγR) were detected in biological fluids from mice and humans. In mouse bearing tumors, circulating amounts of sFcγR increased concurrently with tumor growth. Tumors secreting IgG2a, IgG2b or IgG3 led to a 5- to 10-fold increase in serum sFcγR levels whereas tumors secreting IgG1, IgGA or other types of tumors (non Ig B cell tumors, T cell lymphoma and a melanoma) increased 2- to 3-fold the levels of circulating sFcγR. In the human, sFc7#x03B3;R were also detected in whole unstimulated saliva. Levels of sFcγRII and of sFcγRIII were variable and did not seem to depend on the dental status of the individuals. Finally, a murine recombinant sFcγR (rsFcγR) composed of the two extracellular domains of Fcγ RII was produced by culture of transfected L cells in bioreactors. The purified rsFcγR was found to inhibit antibody production in vitro in anti-SRBC responses and by cultures of small B cells stimulated by anti-IgM antibodies in the presence of IL-4 and IL-5. Moreover, the i.p. injection of this material into adult mice immunized with SRBC led to a decrease of IgG antibody production by splenocytes, as measured by a hemolytic plaque assay, and in serum, as measured by antigen-specific ELISA.

Soluble Fcγ Receptors: Interaction with Ligands and Biological Consequences

Soluble Fcγ receptors are produced by cleavage of the membrane receptors or by alternative splicing. They are found in biologic fluids. After a brief description of the structure and mode of production of soluble FcγR, we address the question of ligands and function of the soluble FcγR by using recombinant molecules and transgenic animals. We show that soluble FcγR are not only IgG-binding factors which interfere with, and block, Fc-dependent immune reactions but also molecules that interact, in vitro, with non-Ig-ligands such as CR3 and CR4 and are trigger or regulate immune functions via these receptors.

N-GLYCAN STRUCTURES OF A RECOMBINANT MOUSE SOLUBLE FCGAMMA RECEPTOR II

N-glycans of a recombinant mouse soluble Fcγ receptor II (sFcγRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226: 139–46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was α-(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and α-(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry.

Recombinant soluble Fcγ receptors: production, purification and biological activities

Abstract Soluble forms of low affinity receptors for the Fc portion of IgG circulate in body fluids and regulate immune functions. We describe the transfection, production and purification techniques which allow the preparation, at a laboratory scale, of milligram amounts of glycosylated recombinant mouse and human soluble Fcγ receptors. These recombinant products bind IgG and are biologically active on immune responses, like their normal counterparts.

Murine soluble Fcγ receptors/IgG-binding factors (IgG-BF): Analysis of the relation to FcγRII and production of milligram quantities of biologically active recombinant IgG-BF

The production of soluble forms of low-affinity FcγR by cells expressing recombinant or natural membrane FcγRII, and the structural relationships between these soluble receptors and membrane FcγRII are described. We show that 37–40 kD soluble FcγRII, corresponding to the two N-terminal domains of FcγRII and binding to IgG, are spontaneously produced in vitro by cleavage of membrane FcγRII. Moreover, we describe methods to produce and purify to homogeneity-large quantities of endotoxin-free recombinant IgG-binding factor (rIgG-BF) from the culture medium of a cell line transfected with a mutated FcγRII cDNA. These methods include the use of bioreactors for culturing transfected fibroblasts and the purification of rIgG-BF by ion-exchange chromatography and hydrophobic-interaction chromatography. By using such procedures, about 2.4 mg of rIgG-BF were purified from 1 liter of culture medium of transfected fibroblasts. Like natural IgG-BF, the 95–99% pure rIgG-BF suppressed, in a dose-dependent manner, secondary in vitro IgG antibody responses to sheep red blood cells.

The carbohydrate moieties of suppressor IgG-binding factor released by murine T cells

The carbohydrate moieties of murine IgG-binding factor (IgG-BF) were studied using lectins binding N-glycosylated sequences such as Concanavalin A (Con A), Lens culinaris agglutinin (LcA), and wheat germ agglutinin (WGA), and lectins binding O-glycosylated sequences such as peanut agglutinin (PNA) and Helix pomatia Agglutinin (HpA). Sources of IgG-BF were: (1) supernatants from T2D4, a T cell hybridoma constitutively producing IgG-BF, and (2) factor purified by affinity chromatography on rabbit IgG-Sepharose, using T2D4 supernatants or supernatants of alloantigen-activated T cells (ATC) as starting material. The presence of IgG-BF was assessed by its ability to inhibit secondary anti-sheep red blood cell (SRBC) IgG antibody responses in vitro and to inhibit rosette formation between Fc gamma receptor (Fc gamma R)-positive spleen cells and erythrocytes sensitized with rabbit anti-Forssman IgG antibodies. Fractionation on immobilized lectins showed that IgG-BF: (1) is completely adsorbed by WGA and PNA and partially by Con A, LcA and HpA, and (2) can be eluted from the five different lectins using the competitor sugars. When produced in the presence of tunicamycin, an inhibitor of N-glycosylation, IgG-BF still binds to HpA which has affinity for O-glycosylated carbohydrate chains. These results indicate that IgG-BF is a glycoprotein with N- and O-glycosylated carbohydrate moieties.