Inulin binding

Abstract: Idiotypes of inulin-binding myeloma proteins (InuBMP) were determined primarly by variable region light chains (VL) or by variable region heavy chains (VH) but needed both chains to be expressed. Recombinant molecules were used to show that individual idiotypes (IdI) of U61, E109, T957, and A4 InuBMP and cross-specific idiotypes (IdXB) of U61 were primarily determined by VL while cross-specific idiotype (IdXA) of A4 was determined mainly by VH. The assignment of genes controlling idiotypes to VH based on allotype linkage (e.g., IdXB) is dubious until the role of the L chain in determining that idiotype is assessed. IdXB has been shown to be a VL-VH marker which presumably is controlled by two unlinked genes. However IdXB can be used as a L chain marker in combinations of strains differing in their L chain genes but having the same permissive H chain genes. Conversely IdXB can be used as a H chain marker in strains having the same permissive L chain genes but differing in their H chain genes.

Abstract: Summary • Recently, the three-dimensional structure of chicory (Cichorium intybus) fructan 1-exohydrolase (1-FEH IIa) in complex with its preferential substrate, 1-kestose, was determined. Unfortunately, no such data could be generated with high degree of polymerization (DP) inulin, despite several soaking and cocrystallization attempts. • Here, site-directed mutagenesis data are presented, supporting the presence of an inulin-binding cleft between the N- and C-terminal domains of 1-FEH IIa. In general, enzymes that are unable to degrade high DP inulins contain an N-glycosylation site probably blocking the cleft. By contrast, inulin-degrading enzymes have an open cleft configuration. • An 1-FEH IIa P294N mutant, introducing an N-glycosylation site near the cleft, showed highly decreased activity against higher DP inulin. The introduction of a glycosyl chain most probably blocks the cleft and prevents inulin binding and degradation. • Besides cell wall invertases, fructan 6-exohydrolases (6-FEHs) also contain a glycosyl chain most probably blocking the cleft. Removal of this glycosyl chain by site-directed mutagenesis in Arabidopsis thaliana cell wall invertase 1 and Beta vulgaris 6-FEH resulted in a strong decrease of enzymatic activities of the mutant proteins. By analogy, glycosylation of 1-FEH IIa affected overall enzyme activity. These data strongly suggest that the presence or absence of a glycosyl chain in the cleft is important for the enzyme's stability and optimal conformation.

Abstract: The entire sequences of the variable region of four heavy chains from BALB/c inulin-binding myeloma proteins have been determined. Among the four proteins there are six amino acid differences, all of which occur in the framework portion of the variable region. All of the six amino acid substitutions can be explained by single base mutations at the DNA level. The pattern of diversity in these proteins is compared to a previously reported group of heavy chains from phosphorylcholine-binding myeloma proteins. Unlike the phosphorylcholine-binding proteins, which (with the exception of two that are identical) have size and sequence differences in their complementarity regions, the inulin-binding heavy chains all have identical complementarity regions with H3 being extremely short. The pattern of variation observed in the anti-inulin heavy chains appears to be most easily explained by a somatic mutation mechanism. However, because none of the substitutions occur in complementarity-determining regions, they presumably would have no selective advantage and would not alter binding specificity. These proteins have further been shown to have crossreacting antigenic determinants (idiotypes). Five of the six sequence differences observed occur at positions that are internal in the molecule and thus presumably would not account for the idiotypic differences. These results suggest that most of the observed idiotypic crossreactivities will be due to differences in the light chains of the anti-inulin proteins.

Abstract: The myeloma proteins binding inulin afford a unique opportunity to study the V region patterns of variation because of the similarity in the VL as well as the VH regions. The diversity patterns in both the VL and VH regions suggest these proteins are encoded by multiple, very similar V gene segments or that somatic mutation may repeatedly generate identical variants. Because of the close similarity in the V domains of these proteins and the extensive idiotypic analyses that have been carried out previously, several interesting conclusions can be drawn about the nature of idiotypic determinants. First, a single amino acid residue may be involved in determining multiple idiotypic determinants. Second, hapten-inhibitable idiotypes may depend on residues within and outside the hypervariable regions. Third, idiotypic similarity does not always predict a corresponding sequence similarity.