scholarly journals Mutational analysis of the carbohydrate-binding activity of the NeuAc(α-2,6)Gal/GalNAc-specific type 2 ribosome-inactivating protein from elderberry (Sambucus nigra) fruits

2002 ◽  
Vol 364 (2) ◽  
pp. 587-592 ◽  
Author(s):  
Ying CHEN ◽  
Pierre ROUGE ◽  
Willy J. PEUMANS ◽  
Els J.M. van DAMME
Keyword(s):  
Mutational Analysis ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Carbohydrate Binding ◽  
Ribosome Inactivating Protein ◽  
Sambucus Nigra ◽  
Original Activity ◽  
Highly Active ◽  
Critical Residue

Sambucus nigra agglutinin I (SNA-I) is a type 2 ribosome-inactivating protein. Site-directed mutagenesis was used to mimic the conversion of the highly active B-chain of fruit-specific SNA (SNA-If) into the completely inactive B-chain of the closely related and naturally occurring loss-of-activity mutant called S. nigra agglutinin lectin-related protein. In the first mutant SNA-If-M1 the high-affinity site 2 of SNA-If was disrupted by replacing the presumed critical residue Asp231 with Glu231. In the double mutant SNA-If-M2, site 1 of SNA-If-M1 was also disrupted by substituting the presumed critical residue Asn48 with Ser48. The parent type 2 ribosome-inactivating protein and both mutants were expressed in Nicotiana tabacum Samsun NN and the recombinant proteins were purified and analysed. Recombinant SNA-If agglutinated rabbit erythrocytes equally well as SNA-If, but both mutants were completely inactive in this test. Binding assays to immobilized galactose and fetuin revealed that the mutation Asp231→Glu231 reduces the affinity of the B-chain for galactose and fetuin by more than 50%. Furthermore, the introduction of the second mutation Asn48→Ser48 reduces the binding activity to less than 20% of the original activity.

scholarly journals Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

1991 ◽  
Vol 11 (9) ◽  
pp. 4356-4362 ◽  
Author(s):  
M N Kanaan ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Mutational Analysis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

scholarly journals Isolation and Molecular Cloning of a Novel Type 2 Ribosome-inactivating Protein with an Inactive B Chain from Elderberry (Sambucus nigra) Bark

1997 ◽  
Vol 272 (13) ◽  
pp. 8353-8360 ◽  
Author(s):  
Els J. M. Van Damme ◽  
Annick Barre ◽  
Pierre Rougé ◽  
Fred Van Leuven ◽  
Willy J. Peumans
Keyword(s):  
Molecular Cloning ◽  
Ribosome Inactivating Protein ◽  
Sambucus Nigra

scholarly journals Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel Crenomytilus grayanus

Marine Drugs ◽  
2018 ◽  
Vol 16 (12) ◽  
pp. 471 ◽  
Author(s):  
Svetlana Kovalchuk ◽  
Nina Buinovskaya ◽  
Galina Likhatskaya ◽  
Valery Rasskazov ◽  
Oksana Son ◽  
...  
Keyword(s):  
Binding Sites ◽  
In Silico Analysis ◽  
Binding Activity ◽  
Cancer Diagnostics ◽  
Site Directed Mutagenesis ◽  
Carbohydrate Binding ◽  
Synthetic Analog ◽  
Crenomytilus Grayanus ◽  
Mussel Crenomytilus Grayanus ◽  
The Individual

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with β-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties.

scholarly journals The major elderberry (Sambucus nigra) fruit protein is a lectin derived from a truncated type 2 ribosome-inactivating protein

1997 ◽  
Vol 12 (6) ◽  
pp. 1251-1260 ◽  
Author(s):  
Els J.M. Damme ◽  
Soma Roy ◽  
Annick Barre ◽  
Pierre Rouge ◽  
Fred Leuven ◽  
...  
Keyword(s):  
Ribosome Inactivating Protein ◽  
Sambucus Nigra

Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa

1991 ◽  
Vol 11 (9) ◽  
pp. 4356-4362
Author(s):  
M N Kanaan ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Mutational Analysis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

scholarly journals Mutational analysis of the carbohydrate binding activity of the tobacco lectin

2010 ◽  
Vol 27 (6) ◽  
pp. 613-623 ◽  
Author(s):  
Dieter Schouppe ◽  
Pierre Rougé ◽  
Yi Lasanajak ◽  
Annick Barre ◽  
David F. Smith ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Binding Activity ◽  
Carbohydrate Binding
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