Binding studies of α-GalNAc-specific lectins to the α-GalNAc (Tn-antigen) form of porcine submaxillary mucin and its smaller fragments

Tarun K. Dam, Thomas A. Gerken, Benildo S. Cavada, Kyria S. Nascimento, Tales R. Moura, Curtis F. Brewer

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Abstract

Isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements demonstrate that a chemically and enzymatically prepared form of porcine submaxillary mucin that possesses a molecular mass of ∼10 6 daltons and ∼2300 α-GalNAc residues (Tn-PSM) binds to the soybean agglutinin (SBA) with a Kd of 0.2 nM, which is ∼10 6-fold enhanced affinity relative to GalNAcα1-O-Ser (Tn), the pancarcinoma carbohydrate antigen. The enzymatically derived 81 amino acid tandem repeat domain of Tn-PSM containing ∼23 α-GalNAc residues binds with ∼103-fold enhanced affinity, while the enzymatically derived 38/40 amino acid cleavage product(s) of Tn-PSM containing ∼11-12 α-GalNAc residues shows ∼102-fold enhanced affinity. A natural carbohydrate decorated form of PSM (Fd-PSM) containing 40% of the core 1 blood group type A tetrasaccharide, and 58% peptide-linked GalNAcα1-O- Ser/Thr residues, with 45% of the peptide-linked α-GalNAc residues linked α-(2,6) to N-glycolylneuraminic acid, shows ∼104 enhanced affinity for SBA. Vatairea macrocarpa lectin (VML), which is also a GalNAc binding lectin, displays a similar pattern of binding to the four forms of PSM, although there are quantitative differences in its affinities as compared with SBA. The higher affinities of SBA and VML for Tn-PSM relative to Fd-PSM indicate the importance of carbohydrate composition and epitope density of mucins on their affinities for lectins. The higher affinities of SBA and VML for Tn-PSM relative to its two shorter chain analogs demonstrate that the length of a mucin polypeptide and hence total carbohydrate valence determines the affinities of the three Tn-PSM analogs. The results suggest a binding model in which lectin molecules "bind and jump" from α-GalNAc residue to α-GalNAc residue along the polypeptide chain of Tn-PSM before dissociating. The complete thermodynamic binding parameters for these mucins including their binding stoichiometries are presented. The results have important implications for the biological activities of mucins including those expressing the Tn cancer antigen.

Original languageEnglish (US)
Pages (from-to)28256-28263
Number of pages8
JournalJournal of Biological Chemistry
Volume282
Issue number38
DOIs
StatePublished - Sep 21 2007

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Mucins
Lectins
Swine
Carbohydrates
Peptides
Antigens
Amino Acids
Tandem Repeat Sequences
Hemagglutination
Molecular mass
Blood Group Antigens
Bioactivity
Titration
Thermodynamics
Stoichiometry
Tn antigen
Epitopes
soybean lectin
Molecules
Chemical analysis

ASJC Scopus subject areas

  • Biochemistry

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Binding studies of α-GalNAc-specific lectins to the α-GalNAc (Tn-antigen) form of porcine submaxillary mucin and its smaller fragments. / Dam, Tarun K.; Gerken, Thomas A.; Cavada, Benildo S.; Nascimento, Kyria S.; Moura, Tales R.; Brewer, Curtis F.

In: Journal of Biological Chemistry, Vol. 282, No. 38, 21.09.2007, p. 28256-28263.

Research output: Contribution to journalArticle

Dam, Tarun K. ; Gerken, Thomas A. ; Cavada, Benildo S. ; Nascimento, Kyria S. ; Moura, Tales R. ; Brewer, Curtis F. / Binding studies of α-GalNAc-specific lectins to the α-GalNAc (Tn-antigen) form of porcine submaxillary mucin and its smaller fragments. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 38. pp. 28256-28263.
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abstract = "Isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements demonstrate that a chemically and enzymatically prepared form of porcine submaxillary mucin that possesses a molecular mass of ∼10 6 daltons and ∼2300 α-GalNAc residues (Tn-PSM) binds to the soybean agglutinin (SBA) with a Kd of 0.2 nM, which is ∼10 6-fold enhanced affinity relative to GalNAcα1-O-Ser (Tn), the pancarcinoma carbohydrate antigen. The enzymatically derived 81 amino acid tandem repeat domain of Tn-PSM containing ∼23 α-GalNAc residues binds with ∼103-fold enhanced affinity, while the enzymatically derived 38/40 amino acid cleavage product(s) of Tn-PSM containing ∼11-12 α-GalNAc residues shows ∼102-fold enhanced affinity. A natural carbohydrate decorated form of PSM (Fd-PSM) containing 40{\%} of the core 1 blood group type A tetrasaccharide, and 58{\%} peptide-linked GalNAcα1-O- Ser/Thr residues, with 45{\%} of the peptide-linked α-GalNAc residues linked α-(2,6) to N-glycolylneuraminic acid, shows ∼104 enhanced affinity for SBA. Vatairea macrocarpa lectin (VML), which is also a GalNAc binding lectin, displays a similar pattern of binding to the four forms of PSM, although there are quantitative differences in its affinities as compared with SBA. The higher affinities of SBA and VML for Tn-PSM relative to Fd-PSM indicate the importance of carbohydrate composition and epitope density of mucins on their affinities for lectins. The higher affinities of SBA and VML for Tn-PSM relative to its two shorter chain analogs demonstrate that the length of a mucin polypeptide and hence total carbohydrate valence determines the affinities of the three Tn-PSM analogs. The results suggest a binding model in which lectin molecules {"}bind and jump{"} from α-GalNAc residue to α-GalNAc residue along the polypeptide chain of Tn-PSM before dissociating. The complete thermodynamic binding parameters for these mucins including their binding stoichiometries are presented. The results have important implications for the biological activities of mucins including those expressing the Tn cancer antigen.",
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AU - Nascimento, Kyria S.

AU - Moura, Tales R.

AU - Brewer, Curtis F.

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N2 - Isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements demonstrate that a chemically and enzymatically prepared form of porcine submaxillary mucin that possesses a molecular mass of ∼10 6 daltons and ∼2300 α-GalNAc residues (Tn-PSM) binds to the soybean agglutinin (SBA) with a Kd of 0.2 nM, which is ∼10 6-fold enhanced affinity relative to GalNAcα1-O-Ser (Tn), the pancarcinoma carbohydrate antigen. The enzymatically derived 81 amino acid tandem repeat domain of Tn-PSM containing ∼23 α-GalNAc residues binds with ∼103-fold enhanced affinity, while the enzymatically derived 38/40 amino acid cleavage product(s) of Tn-PSM containing ∼11-12 α-GalNAc residues shows ∼102-fold enhanced affinity. A natural carbohydrate decorated form of PSM (Fd-PSM) containing 40% of the core 1 blood group type A tetrasaccharide, and 58% peptide-linked GalNAcα1-O- Ser/Thr residues, with 45% of the peptide-linked α-GalNAc residues linked α-(2,6) to N-glycolylneuraminic acid, shows ∼104 enhanced affinity for SBA. Vatairea macrocarpa lectin (VML), which is also a GalNAc binding lectin, displays a similar pattern of binding to the four forms of PSM, although there are quantitative differences in its affinities as compared with SBA. The higher affinities of SBA and VML for Tn-PSM relative to Fd-PSM indicate the importance of carbohydrate composition and epitope density of mucins on their affinities for lectins. The higher affinities of SBA and VML for Tn-PSM relative to its two shorter chain analogs demonstrate that the length of a mucin polypeptide and hence total carbohydrate valence determines the affinities of the three Tn-PSM analogs. The results suggest a binding model in which lectin molecules "bind and jump" from α-GalNAc residue to α-GalNAc residue along the polypeptide chain of Tn-PSM before dissociating. The complete thermodynamic binding parameters for these mucins including their binding stoichiometries are presented. The results have important implications for the biological activities of mucins including those expressing the Tn cancer antigen.

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