Location of sulfhydryl and disulfide groups in bovine .beta.-lactoglobulins and effects of urea

McKenzie, H.A.; Ralston, G.B.; Shaw, Douglas J.

doi:10.1021/bi00774a017

Cited by 122 publications

(72 citation statements)

References 21 publications

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“…Natural BLG was purified from the milk of a single cow homozygous for the variant A of BLG as described by Wal et al (38) and here is considered to be BLG in the native conformation (BLGn). Reduced and carboxymethylated BLG (BLGrcm) was prepared from BLGn as described by Negroni et al (25) by a method slightly modified from that of McKenzie et al (24) and here is considered to be BLG in the denatured conformation (BLGd).…”

Section: Methodsmentioning

confidence: 99%

Induction of Mucosal Immune Response after Intranasal or Oral Inoculation of Mice with Lactococcus lactis Producing Bovine Beta-Lactoglobulin

Châtel

Langella²,

Adel‐Patient

et al. 2001

Clin Diagn Lab Immunol

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The bovine beta-lactoglobulin (BLG) is a major cow's milk allergen. Here, we evaluated the immune response against BLG induced in mice, using the organism Lactococcus lactis, which has GRAS ("generally regarded as safe") status, as a delivery vehicle. The cDNA of the blg gene, encoding BLG, was expressed and engineered for either intra-or extracellular expression in L. lactis. Using a constitutive promoter, the yield of intracellular recombinant BLG (rBLG) was about 20 ng per ml of culture. To increase the quantity of rBLG, the nisin-inducible expression system was used to produce rBLG in the cytoplasmic and extracellular locations. Although the majority of rBLG remained in the cytoplasm, the highest yield (2 g per ml of culture) was obtained with a secreting strain that encodes a fusion between a lactococcal signal peptide and rBLG. Whatever the expression system, the rBLG is produced mostly in a soluble, intracellular, and denatured form. The BLG-producing strains were then administered either orally or intranasally to mice, and the immune response to BLG was examined. Specific anti-BLG immunoglobulin A (IgA) antibodies were detected 3 weeks after the immunization protocol in the feces of mice immunized with the secreting lactococcal strain. Specific anti-BLG IgA detected in mice immunized with lactococci was higher than that obtained in mice immunized with the same quantity of pure BLG. No specific anti-BLG IgE, IgA, IgG1, or IgG2a was detected in sera of mice. These recombinant lactococcal strains constitute good vehicles to induce a mucosal immune response to a model allergen and to better understand the mechanism of allergy induced by BLG.The gastrointestinal tract is constantly exposed to substantial amounts of food and bacterial components. In the healthy gut, the immune system is able to create a balance between mucosal immunity and systemic tolerance. In food allergy, this balance is impaired and oral tolerance to dietary antigen is not achieved or maintained (14,19,29). Cow's milk allergy is an important problem in infants because it affects 1.9 to 2.8% of infants in the first 2 years of life in various countries of northern Europe (17, 18). Beta-lactoglobulin (BLG) is the most abundant protein of the whey fraction of milk and is regarded as a dominant allergen together with casein (37). In our laboratory, we use BLG as a model protein to evaluate and modulate the immune response to a food allergen in mice, and we have produced both anti-BLG monoclonal antibodies (MAb) (25) and recombinant BLG in Escherichia coli (8). Moreover, the BLG structure is well documented (27); it is a 162-aminoacid globular protein which contains two intramolecular disulfide bonds. In this study, we produced BLG in Lactococcus lactis, a food-grade bacterium, and used these recombinant strains to measure a potential specific immune response after intranasal or oral administration in mice.L. lactis is a gram-positive lactic acid bacterium that is nonpathogenic, noninvasive, and noncolonizing and that has GRAS ("generally regar...

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Section: Methodsmentioning

confidence: 99%

Induction of Mucosal Immune Response after Intranasal or Oral Inoculation of Mice with Lactococcus lactis Producing Bovine Beta-Lactoglobulin

Châtel

Langella²,

Adel‐Patient

et al. 2001

Clin Diagn Lab Immunol

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“…A monomer of fJ-Lg contains five cystein residues; one is free, and the others are involved in two intramolecular disulfide bridges. 10 ) In this study, the free sulfhydryl group of the {3-Lg molecule was carboxymethylated (-SH --SCHzCOOH) by a modification of the method used by Yamada et al 11) fJ-Lg (0.45 g) was dissolved in 16 ml of 0.575 M Tris~HCI buffer (pH 8.6) containing 8 M urea and 5.25 mM EDT A. After the solution was degassed, 0.6 g of iodoacetic acid dissolved in 3 ml of I N NaOH was added to the solution.…”

Section: Methodsmentioning

confidence: 99%

Adsorption ofβ-Lactoglobulin onto the Surface of Stainless Steel Particles

Itoh

Nagata

Toyomasu

et al. 1995

Bioscience, Biotechnology, and Biochemistry

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“…The variant Dyak (not shown) isolated by Grosclaude et al (1976) has the substitution Gly for Glu at residue 158. It is assumed that the alternate -SH/-SS positions proposed by McKenzie et al (1972) apply to all variants.…”

Section: Isolation Of Proteinsmentioning

confidence: 99%

Bovine ß-Lactoglobulin E, F and G of Bali (Banteng) Cattle, Bos (Bihos) Javanicus

Bell¹,

Ha²,

Dc³

1981

Aust. Jnl. Of Bio. Sci.

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An electrophoretic examination is made of samples of milk from eight Bali (banteng) cattle, Bos (Bibos) javanicus, in the Northern Territory of Australia. There are two new electrophoretically distinct bovine ft-Iactoglobulins, designated E and F, present in these samples. It is shown by amino acid analysis and tryptic peptide mapping of isolated samples that: (I) both E and F differ from the B variant of domestic cattle (Bos taurus) by + 1 G1y, -1 Glu at residue 157 or 158; and (2) the F variant has the additional charge differences from E and B due to + I Tyr, -1 Asp at residue 129 or 130. It is also shown by studies of peptides produced by the action of Staphylococcus aureus strain V8 protease and by more recent amino acid analyses that (1) the GlyjGlu substitution is at residue 158; (2) there is an additional Bali variant, designated ft-lactoglobulin G, differing from E by + 1 Met, -1 lie at residue 78, but having the same mobility as E; and (3) there is an hitherto undetected neutral residue substitution of + 1 Ser, -1 Pro at position 50 in the F variant. The relation of these variants to other known ft-lactoglobulin variants of the Bos genus is discussed.

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Location of sulfhydryl and disulfide groups in bovine .beta.-lactoglobulins and effects of urea

Abstract: IN /3-lactoglobulin thanks the Engineering Staff of Western Electric Co., Winston-Salem, N. C., for design of the electrical circuitry of the fluorometer. The capable technical assistance of Mrs.Zuzana LaGrange was deeply appreciated.

Cited by 122 publications

References 21 publications

Induction of Mucosal Immune Response after Intranasal or Oral Inoculation of Mice with Lactococcus lactis Producing Bovine Beta-Lactoglobulin

Induction of Mucosal Immune Response after Intranasal or Oral Inoculation of Mice with Lactococcus lactis Producing Bovine Beta-Lactoglobulin

Adsorption ofβ-Lactoglobulin onto the Surface of Stainless Steel Particles

Bovine ß-Lactoglobulin E, F and G of Bali (Banteng) Cattle, Bos (Bihos) Javanicus

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