Monoclonal Antibodies Against Myelin Proteolipid Protein: Identification and Characterization of Two Major Determinants

This report describes the preparation and characterization of a panel of monoclonal antibodies (mAbs) against the myelin proteolipid protein (PLP). A Lewis rat was immunized with bovine proteolipid apoprotein and 27 mAbs were selected based on their reactivity against bovine PLP on enzyme-linked immunosorbent assays. Eleven mAbs recognized the PLP carboxyl-terminal sequence when tested against a panel of synthetic peptides in a solid-phase assay. A carboxyl-terminal pentapeptide (residues 272-276) was sufficient for antibody binding and the terminal phenylalanine residue was found particularly important. Deletion, modification, or replacement of this residue markedly reduced or obliterated antigen-antibody interaction. Nine mAbs reacted with a second antigenic determinant, residues 209-217, but these could be identified only by competitive immunoassays. This peptide was a more effective inhibitor than the longer peptides 202-217 and 205-221, suggesting that flanking residues may interfere with peptide-antibody interaction. Seven antibodies did not react with any of the synthetic peptides tested and their determinants remain unidentified. Immunoblot analysis showed that the mAbs reacted with both the PLP and the DM-20 isoforms. Twenty-three of the mAbs were of the immunoglobulin G2a or b isotype; the remaining antibodies were immunoglobulin M and all of these were specific for residues 209-217. Cultured murine oligodendrocytes were stained by most of the mAbs tested, but the most intense reactivity was observed with the carboxyl-terminus-specific mAbs. The immunocytochemical analyses demonstrate that the mAbs react with the native PLP in situ and show their potential usefulness for studies of the cell biology of myelin and oligodendrocytes.     

Carbohydrate antigens and lectin receptors of the plasma membrane of carrot cells

Summary A monoclonal antibody (JIM 1) has been derived, subsequent to immunization of rats with carrot protoplasts and a hybridoma screen of protoplast immunoagglutination, that recognizes a determinant at the outer face of the plasma membrane of carrot cells. The binding of JIM 1 is readily inhibitable by -D-galactosyl residues. Although weakly cross-reacting with an extracellular arabinogalactan protein, isolated from the conditioned medium of suspension-cultured carrot cells, JIM 1 does not recognize arabinogalactan proteins associated with the plasma membrane. The plasma membrane antigen recognized by JIM 1 was of low molecular weight and was sensitive to both periodate treatment and a protease. JIM 1 therefore defines a new class of galactosyl-residue containing plant cell surface antigen, distinct from the arabinogalactan proteins. However, the extracellular arabinogalactan protein and related plasma membrane-associated glycoproteins are demonstrated to bind the anti-galactose plant lectin peanut agglutinin.Abbrevations AGP arabinogalactan protein - McAb monoclonal antibody - PNA peanut agglutinin     

Molecular evolution and subunit structure of cattle lens alpha crystallin

Summary The present studies were based on the premise that any common determinants in homologous proteins must have originated with the common ancestor of all of the taxonomic groups in which that determinant occurs. Cross-reacting antigenic determinants of lens alpha crystallin in various classes of modern vertebrates were used to trace their evolutionary relationships.For quantitation of evolutionarily distinct determinants, equimolar amounts of alpha crystallin or its subunits, in either monomeric or reaggregated form, were bound to a matrix, then saturated with^{1 2 5}I-labeled Fab fragments of anti-cattle alpha crystallin antibodies having phylogenetically restricted specificities. This quantitative procedure has the important advantage of independence from variation in antibody responses to different determinants of the same antigenic molecule. The procedure is not impaired by steric hindrance.Both the SH-containing and SH-free subunits of cattle lens alpha crystallin were found to contain common antigenic determinants with the cyclostomata alpha crystallin. Such determinants originated in evolution with the first vertebrates, the primitive agnatha. Antigenic determinants transferred from ancestral aquatic and land vertebrates to the mammals were found to constitute 93% of all determinants reactive in the monomeric SH-free subunits of cattle alpha crystallin. These determinants constitute only 76.5% of all determinants which are reactive in the SH-containing subunits. The antigenic determinants on both types of subunits were all found to be different. These findings indicate that evolutionary changes must have occurred more slowly in SH-free subunits than in SH-containing subunits.Significant decreases or increases were found in the content of various evolutionarily distinct determinants reactive in the reaggregated subunits as compared to the ones reactive in monomeric subunits. These differences can result from the formation of new conformational antigenic determinants during aggregation as well as from the burial or exposure of other determinants after aggregation.Different amounts of evolutionarily distinct antigenic determinants were found to be reactive in the molecules dissociated into subunits than in the intact molecules one of the reasons being that the intact molecules contain phylogenetically distinct determinants which depend on the quaternary structure of the protein molecule. The data obtained indicate that the quaternary structure of cattle alpha crystallin has, to a large degree, remained unchanged since the origin of vertebrates.     

Seasonal succession,standing crop and determinants of primary productivity of the phytoplankton of Ministik Lake,Alberta, Canada

Ministik Lake (longitude 113°01; latitude 53°21), a well-mixed, shallow (mean depth 1.83 m), eutrophic lake is characterized by eutrophic chlorococcalean and cyanophycean phytoplankton associations, and little change in standing crop size with increasing depth. Standing crops and primary productivity are low during the winter but pronounced spring and late summer/early autumn maxima occur. Mean yearly areal standing crop (B) and primary productivity (A) were 199.2 mg m^–2 chlorophyll a and 319.5 mg C hr^–1 m^–2 respectively. Annual productivity was estimated at 1399,6 g C m^–2yr^–1. The mean increase in the extinction coefficient () per unit increase in standing crop (B) was 0.03 In units m^–1. High non-algal light attenuation (q) occurred averaging 46% which prevented the ratio B from attaining more than 74.2% of the theoretical maximum except once when selfshading occurred. Insignificant relationships existed between B (mg m^–3 chlorophyll a) and A_max (mg C hr^–1 m^–3), A and B and A and B. Close correlations existed between A and A_max/ and A and I₀ (W m^–2). The depth of the euphotic zone (Z_eu) varied between 0.6 and 2.0 m; the average relationship between z_eu and was z_eu = 3.78/_te, and the mean standing crop in the euphotic zone represented 58.3% of the theoritical maximum. The high _q values made the model of Talling (1957) inapplicable to this lake. The Q₁₀ value for the lake was 1.20. _max (mg C chlorophyll a ^–1 hr^–1) was closely related to both temperature and irradiance, and depressed by high pH values.Growth of the cyanophycean algae was correlated with temperature, chlorophycean algae with phosphate-phosphorus and temperature and the diatoms with dissolved silica and phosphate-phosphorus, but only in the case of Chaetoceros elmorii did any nutrient appear limiting. Indirect evidence that free CO₂ limited photosynthetic rates is provided by the _max:pH relationship.     

Probing the sweet determinants of brazzein: wild-type brazzein and a tasteless variant, brazzein-ins(R18a-I18b), exhibit different pH-dependent NMR chemical shifts

Brazzein is a small, intensely sweet protein. As a probe of the functional properties of its solvent-exposed loop, two residues (Arg-Ile) were inserted between Leu18 and Ala19 of brazzein. Psychophysical testing demonstrated that this mutant is totally tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that residues affected by the insertion are localized to the mutated loop, the region of the single alpha-helix, and around the Cys16-Cys37 disulfide bond. Residues unaffected by this mutation included those near the C-terminus and in the loop connecting the alpha-helix and the second beta-strand. In particular, several residues of brazzein previously shown to be essential for its sweetness (His31, Arg33, Glu41, Arg43, Asp50, and Tyr54) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His31, Glu41, Asp50, and Tyr54 were unaltered by the insertion. The insertion led to large chemical shift and pKa perturbation of Glu36, a residue shown previously to be important for brazzein's sweetness. These results serve to refine the known sweetness determinants of brazzein and lend further support to the idea that the protein interacts with a sweet-taste receptor through a multi-site interaction mechanism, as has been postulated for brazzein and other sweet proteins (monellin and thaumatin).     

Unraveling a hotspot for TCR recognition on HLA-A2: evidence against the existence of peptide-independent TCR binding determinants

T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 alpha1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.     

Identification of polypeptides specific to rachis abscission zone cells of Sambucus nigra

Specific polypeptides and antigenic determinants in abscission zone cells of the leaf rachis of Sambucus nigra L. (elderberry) were identified. Extracts from abscission zone tissue (OZ) and from ethylene-treated abscission zone tissue after separation (Zone) were probed, using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological techniques, for unique peptide components absent from neighbouring non-zone mid-rachis tissue (MR). Ouchterlony immuno-diffusion revealed differences in the spectrum of antigenic determinants possessed by each tissue type when challenged with antiserum raised against OZ. Immuno-electrophoresis showed a basic polypeptide which is expressed preferentially in the abscission zone.
Immune-competition of OZ, MR and Zone extracts using immuno-affinity column chromatography has identified polypeptides of ca 34, ca 32, and ca 28 kDa which are (up to the limits of detection) abscission-cell specific. An antibody raised against the ca 34 kDa polypeptide recognises a peptide of ca 34 kDa present in OZ and Zone. This peptide is absent from MR.
These results suggest that the specific positional differentiation of ethylene-responsive target cells which constitute the leaf rachis abscission zone in S. nigra is accompanied by the expression of zone-cell-specific antigenic determinants which are not expressed by non-target neighbouring tissue.     

Differences in the amino acid distributions of 3(10)-helices and alpha-helices.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.