Immunological Cross-Reactions of alpha-Lactalbumins from Different Species

Prieels, Jean‐Paul; Poortmans, Jacques; Dolmans, Marcel; Léonis, J

doi:10.1111/j.1432-1033.1975.tb09892.x

Cited by 22 publications

(7 citation statements)

References 9 publications

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“…The experimental system resolves nonprecipitating antigen-antibody complexes as well as precipitating ones. The importance of recognizing nonprecipitating complexes has been previously illustrated by work on cytochromes c (25) and a lactalbumins (26). This resolution is essential since purified tubulin elicits only poorly precipitating or nonprecipitating antibodies in our experience.…”

Section: Methodsmentioning

confidence: 96%

Species-dependent immunological differences between vertebrate brain tubulins.

Morgan¹,

Holladay²,

Spooner³

1978

Proc. Natl. Acad. Sci. U.S.A.

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The antigenic similarities and differences between highly purified brain tubulins from lamb, mouse, and chick embryo have been examined using rabbit antisera prepared against each of these tubulins. These antisera are capable of binding l25I-labeled tubulin in homologous or heterologous combinations, demonstrating immunological similarity between the tubulins. However ences between tubulins from different classes of microtubules and species differences between flagellar outer doublet tubulins.The present study has quantitatively analyzed the purified cytoplasmic brain tubulins from three different vertebrate species by radioimmunoassay. The results demonstrate that although heterologous combinations of tubulin and antisera will interact, there are clear quantitative differences in the ability of the various radiolabeled tubulins to bind to the antibodies and in the ability of the various unlabeled tubulins to compete with the different labeled tubulins for antibody binding sites. MATERIALS AND METHODSTubulin Purification. Tubulin was isolated and purified from lamb brain, mouse brain, and 14-day embryo chick brain by two cycles of polymerization in 0.1 M 1,4-piperazinediethanesulfonic acid (Pipes) (pH 6.8), containing 1 mM GTP, 1 mM MgCl2, 0.5 mM ethylene glycol bis(f3-aminoethyl ether)-N,N'-tetraacetate (EGTA), and 50% glycerol, followed by chromatography on a column of phosphocellulose (14, 15). Purity was assessed by electrophoresis on sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gels (16).Immunization and Antigen Todination. Antisera to Iamb, mouse, and chick brain tubulins were produced by immunizing rabbits with purified antigen that had been partially crosslinked with glutaraldehyde (17). Rabbits were injected subcutaneously with 2 mg of antigen in complete Freund's adjuvant, followed at 2-week intervals by boosters in incomplete Freund's adjuvant. Each antiserum discussed in this paper was collected from a single rabbit.Purified tubulins were radiolabeled with 125I through the iodinated imidoester, methyl-3,5-diiodo-p-hydroxybenzimidate, by a modification of the technique of Wood et al. (18). Ten microliters of a 20 mM solution of methyl-p-hydroxybenzimidate-HCl in 50 mM sodium borate (pH 8.5) was mixed with 10 ,l of a 40mM chloramine T solution, 10 1.l of a 40mM NaI solution, and 10 0A of 125I (-1 mCi) and allowed to react at room temperature for 15 min. The reaction was stopped by the addition of 1 ,l of 1 M 2-mercaptoethanol, and the iodinated imidoester was precipitated by neutralizing with 1 M acetic acid (-5 ,l). The iodinated imidoester was collected by centrifugation at 10,000 X g for 5 min, and the pellet was dissolved in 25 til of 50 mM sodium borate (pH 8.5). Approximately 25 ,4g of purified tubulin was added to the iodinated imidoester, and the mixture was reacted for 24 hr at room temperature. Unbound iodinated imidoester was then removed from the solution by dialysis against borate-buffered saline (pH 8.0), and the iodinated protein was diluted to 10 ml with borate-buffered sali...

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Section: Methodsmentioning

confidence: 96%

Species-dependent immunological differences between vertebrate brain tubulins.

Morgan¹,

Holladay²,

Spooner³

1978

Proc. Natl. Acad. Sci. U.S.A.

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“…The aim of the present study was to demonstrate whether cartilage proteoglycans from different species and locations cross-react immunologically, in analogy with many other proteins (Arons & Neurath, 1969;Pricels et al, 1975).…”

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confidence: 98%

Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species

Wieslander¹

1981

Biochemical Journal

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Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.

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“…There has been additional evidence to support the existence of unique conformational homologies directly amongst the differing a-lactalbumin species by virtue of their cross-reactivity with galactosyltransferases of other species (Ley & Jenness, 1970; Quarfoth & Jenness, 1975;Hopper & McKenzie, 1974). On the other hand, immunological cross-reactivity between various -lactalbumins has been ambiguous and perhaps has not shown as much homology, especially between evolutionarily distant species (Tanahashi et al, 1968;Prieels et al, 1975). The low levels of cross-reactivity between some species imply subtle differences in surface determinants for antigenic specificity (Tanahashi et al, 1968;Prieels, et al, 1975).…”

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confidence: 99%

Nuclear magnetic resonance characterization of aromatic residues of .alpha.-lactalbumins. Laser photochemically induced dynamic nuclear polarization nuclear magnetic resonance studies of surface exposure

Berliner¹,

Kaptein²

1981

Biochemistry

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The alpha-lactalbumins are involved as modifier proteins in lactose biosynthesis. Lactalbumins from different mammalian species are cross-reactive with the galactosyltransferases from others because of their homologous primary and tertiary structure. We have studied the surface exposure of several alpha-lactalbumins (bovine, goat, human, guinea pig, and rabbit) by the laser photo-CIDNP technique, an NMR method which measures the access of a photoexcited flavin dye to surface exposed Tyr, Trp, and His residues. An exposed histidine-68 residue exists in the bovine and goat species but is missing in other species and is replaced with a His-10 in the guinea pig species. The only exposed tryptophan residue was Trp-104, which cross-relaxed to nearby Trp-60. Cross-polarization to the latter residue was proven by taking free induction decays after extremely short light pulses (0.1 s). Furthermore, this cross-polarized Trp-60 resonance was absent in the guinea pig species, which has a substitution at position 60. The chemical shifts of these Trp residues were extremely similar in all species but rabbit, which has substituted His for a Tyr at position 103, situated close to both Trp-104 and -60. Three of the four tyrosines in bovine, goat, human, and guinea pig alpha-lactalbumin were exposed (the latter species has an extra Tyr in its sequence which was apparently not accessible). The rabbit species contains only two Tyr residues, the one which was not accessible (Tyr-50) being common to all species.

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Immunological Cross-Reactions of alpha-Lactalbumins from Different Species

Cited by 22 publications

References 9 publications

Species-dependent immunological differences between vertebrate brain tubulins.

Species-dependent immunological differences between vertebrate brain tubulins.

Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species

Nuclear magnetic resonance characterization of aromatic residues of .alpha.-lactalbumins. Laser photochemically induced dynamic nuclear polarization nuclear magnetic resonance studies of surface exposure

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