Limits...
Mutations in the human lambda5/14.1 gene result in B cell deficiency and agammaglobulinemia.

Minegishi Y, Coustan-Smith E, Wang YH, Cooper MD, Campana D, Conley ME - J. Exp. Med. (1998)

Bottom Line: The three substitutions correspond to the sequence in the lambda5/14. 1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline.When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant lambda5/14.1.These findings indicate that expression of the functional lambda5/14.1 is critical for B cell development in the human.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennesse 38105, USA.

ABSTRACT
B cell precursors transiently express a pre-B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of lambda5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igalpha and Igbeta. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine lambda5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for lambda5/14.1. The maternal allele carried a premature stop codon in the first exon of lambda5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the lambda5/14. 1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant lambda5/14.1. These findings indicate that expression of the functional lambda5/14.1 is critical for B cell development in the human.

Show MeSH

Related in: MedlinePlus

Transfection of  COS7 cells with expression vectors for normal or mutant λ5/ 14.1 and normal VpreB. (A)  Cells were transfected with VpreB  and normal λ5/14.1 (lane 1),  VpreB and mutant λ5/14.1 (lane  2), VpreB alone (lane 3), or an  empty vector (lane 4). After  metabolic labeling, both lysates  and supernatants were immunoprecipitated with antibody to  VpreB. (B) COS7 cells were transfected with normal λ5/14.1 (lane  1), mutant λ5/14.1 (lane 2), or  an empty vector (lane 3), immunoprecipitated using a polyclonal  antilambda antibody, and electrophoresed under reducing (R)  or nonreducing (NR) conditions.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199185&req=5

Figure 5: Transfection of COS7 cells with expression vectors for normal or mutant λ5/ 14.1 and normal VpreB. (A) Cells were transfected with VpreB and normal λ5/14.1 (lane 1), VpreB and mutant λ5/14.1 (lane 2), VpreB alone (lane 3), or an empty vector (lane 4). After metabolic labeling, both lysates and supernatants were immunoprecipitated with antibody to VpreB. (B) COS7 cells were transfected with normal λ5/14.1 (lane 1), mutant λ5/14.1 (lane 2), or an empty vector (lane 3), immunoprecipitated using a polyclonal antilambda antibody, and electrophoresed under reducing (R) or nonreducing (NR) conditions.

Mentions: To determine the functional consequences of the amino acid substitution in exon 3 of λ5/14.1, expression vectors for VpreB and the normal and mutant λ5/14.1 genes were introduced into COS7 cells by lipofection. In metabolically labeled cells transfected with VpreB and the normal or mutant λ5/14.1, both VpreB and λ5/14.1 could be immunoprecipitated from cell lysates using a monoclonal anti-VpreB antibody, demonstrating that the mutant as well as the normal λ5/14.1 could bind to VpreB. However, analysis of supernatants from cells transfected with the same vectors indicated that normal λ5/14.1 was secreted into the supernatant with VpreB, as previously described (13); whereas the mutant λ5/14.1 was not secreted (Fig. 5 A).


Mutations in the human lambda5/14.1 gene result in B cell deficiency and agammaglobulinemia.

Minegishi Y, Coustan-Smith E, Wang YH, Cooper MD, Campana D, Conley ME - J. Exp. Med. (1998)

Transfection of  COS7 cells with expression vectors for normal or mutant λ5/ 14.1 and normal VpreB. (A)  Cells were transfected with VpreB  and normal λ5/14.1 (lane 1),  VpreB and mutant λ5/14.1 (lane  2), VpreB alone (lane 3), or an  empty vector (lane 4). After  metabolic labeling, both lysates  and supernatants were immunoprecipitated with antibody to  VpreB. (B) COS7 cells were transfected with normal λ5/14.1 (lane  1), mutant λ5/14.1 (lane 2), or  an empty vector (lane 3), immunoprecipitated using a polyclonal  antilambda antibody, and electrophoresed under reducing (R)  or nonreducing (NR) conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199185&req=5

Figure 5: Transfection of COS7 cells with expression vectors for normal or mutant λ5/ 14.1 and normal VpreB. (A) Cells were transfected with VpreB and normal λ5/14.1 (lane 1), VpreB and mutant λ5/14.1 (lane 2), VpreB alone (lane 3), or an empty vector (lane 4). After metabolic labeling, both lysates and supernatants were immunoprecipitated with antibody to VpreB. (B) COS7 cells were transfected with normal λ5/14.1 (lane 1), mutant λ5/14.1 (lane 2), or an empty vector (lane 3), immunoprecipitated using a polyclonal antilambda antibody, and electrophoresed under reducing (R) or nonreducing (NR) conditions.
Mentions: To determine the functional consequences of the amino acid substitution in exon 3 of λ5/14.1, expression vectors for VpreB and the normal and mutant λ5/14.1 genes were introduced into COS7 cells by lipofection. In metabolically labeled cells transfected with VpreB and the normal or mutant λ5/14.1, both VpreB and λ5/14.1 could be immunoprecipitated from cell lysates using a monoclonal anti-VpreB antibody, demonstrating that the mutant as well as the normal λ5/14.1 could bind to VpreB. However, analysis of supernatants from cells transfected with the same vectors indicated that normal λ5/14.1 was secreted into the supernatant with VpreB, as previously described (13); whereas the mutant λ5/14.1 was not secreted (Fig. 5 A).

Bottom Line: The three substitutions correspond to the sequence in the lambda5/14. 1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline.When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant lambda5/14.1.These findings indicate that expression of the functional lambda5/14.1 is critical for B cell development in the human.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennesse 38105, USA.

ABSTRACT
B cell precursors transiently express a pre-B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of lambda5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igalpha and Igbeta. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine lambda5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for lambda5/14.1. The maternal allele carried a premature stop codon in the first exon of lambda5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the lambda5/14. 1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant lambda5/14.1. These findings indicate that expression of the functional lambda5/14.1 is critical for B cell development in the human.

Show MeSH
Related in: MedlinePlus