Remarkable selective glycosylation of the immunoglobulin variable region in follicular lymphoma.

Follicular lymphoma (FL) generally expresses immunoglobulin (Ig) with somatically mutated variable (V) region genes. Surprisingly, these almost always carry introduced motifs available for N-glycosylation (Asn-X-Ser/Thr). Introduced motifs are uncommon on normal B cells, but are on other germinal center (GC)-associated B-cell malignancies suggesting a site-specific role. They are not evident in mutated chronic lymphocytic leukemia (CLL) or myeloma. Recently, we found that the glycosylation sites are unusual in containing oligomannose glycans, which are apparently displayed on tumor cell surface IgM. This suggests a potential interaction with a mannose receptor in the GC. However, natural N-glycosylation sites exist in germline (GL) V region genes, particularly the V4-34 gene expressed by normal B cells and by some malignancies, including CLL, potentially undermining the selective importance for FL. To compare oligosaccharide addition at the introduced and natural sites, we expressed V region genes as single chain Fv (scFv) and analyzed the added glycans. In contrast to introduced sites, which were oligomannosylated, the natural GL motif in the V4-34 sequence had no added sugars. The remarkable selective glycosylation within the heavy chain V region gene of FL apparently permits only limited processing to oligomannose at somatically mutated motifs, creating a feature exploitable by GC lymphomas.

Biased use of VH5 IgE-positive B cells in the nasal mucosa in allergic rhinitis.

BACKGROUND: IgE antibody-producing B cells are enriched in the nasal mucosa in patients with allergic rhinitis because of local class switching to IgE. The expressed IgE VH genes also undergo somatic hypermutation in situ to generate clonal families. The antigenic driving force behind these events is unknown. OBJECTIVE: To examine the possible involvement of a superantigen in allergic rhinitis, we compared the variable (VH) gene use and patterns of somatic mutation in the expressed IgE heavy-chain genes in nasal biopsy specimens and blood from allergic patients and the IgA VH use in the same biopsy specimens and also those from nonallergic controls. METHODS: We extracted mRNA from the nasal biopsy specimens of 13 patients and 4 nonallergic control subjects and PBMCs from 7 allergic patients. IgE and IgA VH regions were RT-PCR amplified, and the DNA sequences were compared with those of control subjects. We constructed a molecular model of VH5 to locate amino acids of interest. RESULTS: We observed a significantly increased frequency of IgE and IgA VH5 transcripts in the nasal mucosa of the allergic patients compared with the normal PBMC repertoire. Within IgE and IgA VH5 sequences in the nasal mucosa, the distribution of replacement amino acids was skewed toward the immunoglobulin framework regions. Three of 4 nonintrinsic hotspots of mutation identified in the VH5 sequences were in framework region 1. The hotspots and a conserved VH5-specific framework residue form a tight cluster on the surface of VH5. CONCLUSION: Our results provide evidence for the activity of a superantigen in the nasal mucosa in patients with allergic rhinitis.

Key residues contributing to dominant conformational autoantigenic epitopes on thyroid peroxidase identified by mutagenesis.

Thyroid peroxidase (TPO) is a major autoantigen in thyroid autoimmune disease where pathogenic autoantibodies recognise conformational epitopes restricted to two overlapping immunodominant regions (IDR) termed IDR-A and -B. Based upon our structural model of TPO, we report on the localisation of the IDRs to specific amino acids important for autoantibody binding. Using a panel of recombinant human Fabs (rhFabs) from autoimmune patients, specific for the IDR-A or -B epitopes, in combination with eukaryotic expression of 14 single amino acid mutants of TPO, we identify R225 and K627 as key components of IDR-A and -B, respectively. Moreover, each mutant specifically led to the loss of binding of three different IDR-A- or -B-specific rhFabs, without affecting the binding of autoantibodies to the other determinant. Further supportive evidence for the role of amino acids R225 and K627 was obtained with murine monoclonal antibodies that first defined the IDRs. The identification of amino acids R225 and K627 as key residues for the IDR epitopes on TPO will advance our understanding of the molecular basis of autoreactivity and facilitate the design of novel therapeutic agents.

Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen.

The monoclonal IgM cold agglutinins that bind to the I/i carbohydrate Ags on the surface of RBCs all have Ig H chains encoded by the V4-34 gene segment. This mandatory use indicates that distinctive amino acid sequences may be involved in recognition. Critical amino acids exist in framework region 1 (FR1) of V4-34-encoded Ig, and these generate a specific Id determinant which apparently lies close to the I binding site. However, I binding by Id-expressing Ig can be modulated by sequences in complementarity-determining region (CDR)(H)3. Examination of the crystal structure of an anti-I cold agglutinin has revealed a hydrophobic patch in FR1 involving residue W7 on beta-strand A and the AVY motif (residues 23-25) on beta-strand B. In this study we used mutagenesis to show that each of the strand components of the hydrophobic patch is required for binding the I carbohydrate Ag. In addition, the crystal structure reveals that amino acids in the carboxyl-terminal region of CDR(H)3 form a surface region adjacent to the hydrophobic patch. We propose that the I carbohydrate Ag interacts simultaneously with the entire hydrophobic patch in FR1 and with the outside surface of CDR(H)3. This interaction could leave most of the conventional binding site available for binding other Ags.

A novel immunoglobulin superfamily receptor (19A) related to CD2 is expressed on activated lymphocytes and promotes homotypic B-cell adhesion.

A novel lymphocyte-specific immunoglobulin superfamily protein (19A) has been cloned. The predicted 335-amino-acid sequence of 19A represents a Type 1 membrane protein with homology with the CD2 family of receptors. A molecular model of the two predicted extracellular immunoglobulin-like domains of 19A has been generated using the crystal structure of CD2 as a template. In isolated lymphocytes, expression of 19A is induced by various activation stimuli, and enforced expression of the 19A gene promotes homotypic cell adhesion in a B-cell-line model. Collectively these data imply that the 19A protein plays a role in regulation of lymphocyte adhesion.