Structure and function in IgA.
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Animals↗
Biomedical subjects
L Carayannopoulos
Publications and source records attributed to L Carayannopoulos.
Localization of the binding site for the monocyte immunoglobulin (Ig) A-Fc receptor (CD89) to the domain boundary between Calpha2 and Calpha3 in human IgA1.
Immunoglobulin (Ig) A serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities of blood. In playing its role in humoral immunity, IgA interacts with a variety of effector molecules present both in serum and on the surfaces of immune and inflammatory cells. To study these interactions, we previously established expression of human IgA1 in insect cells using recombinant baculoviruses and showed that the expressed antibody is a structurally and functionally intact polypeptide useful for examining the molecular properties of IgA. Indeed, since the C alpha 2 N-linked glycosylation site lies near the Fab-distal pole of C alpha 2, the inability of a mutant IgA1 lacking C alpha 2 N-glycosylation to bind its cognate receptor suggested that the monocyte Fc alpha receptor (mFcalphaR) recognizes IgA at a hinge-distal site encompassing the boundary between the C alpha 2 and C alpha 3 domains. In this report, we utilize both domain-swapped IgA/IgG and point-mutated IgA chimeras to verify the above hypothesis. Using an antigen-specific rosetting assay and a mFc alpha R-expressing cell line, we show that (a) C alpha 2 and C alpha 3 together are necessary and sufficient for binding; (b) neither the IgA hinge nor the tailpiece is necessary for binding; (c) mutations away from the interdomain boundary do not affect binding; and (d) mutations located near the three-dimensional boundary between C alpha 2 and C alpha 3 completely disrupt binding. Taken together, these results localize the mFc alpha R recognition site on IgA to the boundary region between the second and third constant domains--a site analogous to that recognized by Staphylococcus aureus protein A on IgG. The use of this hinge-distal site is, to date, unique among Fc receptors of the Ig superfamily.
Amino Acid Sequence↗
Recombinant human IgA expressed in insect cells.
IgA serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities in serum. To map the sites of interaction of immune effector molecules with the IgA constant region (C alpha), we have expressed soluble, chimeric human IgA in insect cells using recombinant baculoviruses. This antibody is correctly assembled into heavy chain/light chain heterodimers, N-glycosylated, and secreted by the insect cells; further, when coexpressed with a human J chain, the antibodies can assemble into dimers. The recombinant protein is authentic by a number of criteria, including antigen-binding, recognition by monoclonal antibodies, complement fixation via the alternative pathway, and specific binding to the monocyte IgA Fc receptor. We have also constructed viruses which encode structurally altered IgA heavy chains. Using one of these variant viruses, we have shown that glycosylation of the second domain of C alpha is required for interaction with the monocyte IgA Fc receptor. This system should prove useful in further characterization of the structure-function relationships in human C alpha.
Animals↗
NonO, a non-POU-domain-containing, octamer-binding protein, is the mammalian homolog of Drosophila nonAdiss.
We have cloned the ubiquitous form of an octamer-binding, 60-kDa protein (NonO) that appears to be the mammalian equivalent of the Drosophila visual and courtship song behavior protein, no-on-transient A/dissonance (nonAdiss). A region unprecedently rich in aromatic amino acids containing two ribonuclear protein binding motifs is highly conserved between the two proteins. A ubiquitous form of NonO is present in all adult tissues, whereas lymphocytes and retina express unique forms of NonO mRNA. The ubiquitous form contains a potential helix-turn-helix motif followed by a highly charged region but differs from prototypic octamer-binding factors by lacking the POU DNA-binding domain. In addition to its conventional octamer duplex-binding, NonO binds single-stranded DNA and RNA at a site independent of the duplex site.
Amino Acid Sequence↗
Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein.
Monoclonal antibodies MOv18 and MOv19, raised against a membrane preparation of an ovarian carcinoma surgical specimen, react with a surface antigen present on the majority of nonmucinous ovarian malignant tumors tested but not with normal adult tissue (S. Miotti, S. Canevari, S. Ménard, D. Mezzanzanica, G. Porro, S. M. Pupa, M. Regazzoni, E. Tagliabue, and M. I. Colnaghi, Int. J. Cancer, 39: 297-303, 1987). This surface antigen was purified as a soluble glycoprotein (molecular mass, 36-38 kDa) released from the cell surface of an ovarian carcinoma cell line (IGROV1) by digestion with Bacillus thuringiensis phospholipase C. Immunoblotting demonstrated that the purified protein reacted with MOv18 and MOv19 and that treatment of the purified preparation with N-glycanase resulted in a protein with a molecular mass of 27 kDa. The NH3-terminal amino acid sequence of the purified antigen was determined. This sequence is highly homologous to an internal stretch of 27 amino acids located near the NH3 terminus of human folate-binding protein. An oligonucleotide probe was synthesized and used to screen an IGROV1 ovarian carcinoma, lambda gt11 complementary DNA library to obtain three complementary DNA clones. The complete nucleotide sequence of one of these complementary DNA clones was determined. This sequence is nearly identical to that of a folate-binding protein clone obtained from the Caco-2 human carcinoma cell line. In addition, the nucleotide sequence of the 5'-untranslated region of the other two clones was determined. This region of all three clones was different. The product of the Caco-2 folate-binding protein clone expressed in Chinese hamster ovary cells was recognized by the MOv18 and MOv19 antibodies, confirming that the antigen and folate-binding protein are one and the same. Furthermore, a cell line that binds the MOv18 and MOv19 antibodies expressed increased levels of folate-binding protein mRNA compared with a cell line that does not bind these antibodies. These results indicate that the MOv18 and MOv19 monoclonal antibodies bind to at least one form of folate-binding protein and that this protein, which is evidently overexpressed in certain malignant tumors, may provide a suitable target for immunotherapy with these antibodies.
Amino Acid Sequence↗
The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes.
All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression.
Base Sequence↗