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Mapping the Binding Site of a Cross-Reactive Plasmodium falciparum PfEMP1 Monoclonal Antibody Inhibitory of ICAM-1 Binding.

Lennartz F, Bengtsson A, Olsen RW, Joergensen L, Brown A, Remy L, Man P, Forest E, Barfod LK, Adams Y, Higgins MK, Jensen AT - J. Immunol. (2015)

Bottom Line: When mapped onto a homology model of DBLβ3_D4, these cluster to a defined, surface-exposed region on the convex surface of DBLβ3_D4.Mutagenesis confirmed that the site most strongly protected is necessary for 24E9 binding, which is consistent with a low-resolution structure of the DBLβ3_D4::24E9 Fab complex derived from small-angle x-ray scattering.The convex surface of DBLβ3_D4 has previously been shown to contain the ICAM-1 binding site of DBLβ domains, suggesting that the mAb acts by occluding the ICAM-1 binding surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;

No MeSH data available.


Related in: MedlinePlus

24E9 mAb recognizes a conformation epitope on PFD1235w DBLβ3_D4. (A) Western blotting of PFD1235w DBLβ3_D4 (D4) and DBLβ3_D5 (D5). +DTT (reduced), −DTT (nonreduced). Lane 1, Prosieve protein marker (M) visualized by phosphorescent paint as dots. Lanes 2 and 3, DBLβ_D4 (±DTT). Lanes 4 and 5, DBLβ3_D5 (±DTT). Arrow shows nonreduced DBLβ3_D4 (lane 3) recognized by 24E9 mAb. (B) 24E9 mAb ELISA reactivity against reduced (+DTT) and nonreduced (−DTT) PFD1235w DBLβ3_D4. (C) AB01 mAb ELISA reactivity against reduced (+DTT) and nonreduced (−DTT) DBLγ of PFD1235w. Mean OD values are shown for three independent experiments. Error bars indicate SD.
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fig05: 24E9 mAb recognizes a conformation epitope on PFD1235w DBLβ3_D4. (A) Western blotting of PFD1235w DBLβ3_D4 (D4) and DBLβ3_D5 (D5). +DTT (reduced), −DTT (nonreduced). Lane 1, Prosieve protein marker (M) visualized by phosphorescent paint as dots. Lanes 2 and 3, DBLβ_D4 (±DTT). Lanes 4 and 5, DBLβ3_D5 (±DTT). Arrow shows nonreduced DBLβ3_D4 (lane 3) recognized by 24E9 mAb. (B) 24E9 mAb ELISA reactivity against reduced (+DTT) and nonreduced (−DTT) PFD1235w DBLβ3_D4. (C) AB01 mAb ELISA reactivity against reduced (+DTT) and nonreduced (−DTT) DBLγ of PFD1235w. Mean OD values are shown for three independent experiments. Error bars indicate SD.

Mentions: To determine whether 24E9 mAb interacts with a conformational epitope, we used Western blotting to test the reactivity of 24E9 mAb to reduced and nonreduced DBLβ3_D4. As a control, we performed the same experiment with the non-DC4 PFD1235w DBLβ3_D5 domain. 24E9 recognized only nonreduced DBLβ3_D4 (Fig. 5A). We observed the same result by ELISA (Fig. 5B), where 24E9 recognized only nonreduced DBLβ3_D4. To test whether the loss of reactivity of the mAb toward DTT-treated PFD1235W DBLβ3_D4 was a result of the mAb being reduced in the ELISA, we performed the same assay using the PFD1235w DBLγ-specific human mAb (AB01), which is only partially dependent on the correct folding of DBLγ (24). AB01 mAb was still able to recognize DTT-treated DBLγ (Fig. 5C) showing that a similar mAb remained intact in the ELISA. This suggests that 24E9 mAb targets a conformational epitope.


Mapping the Binding Site of a Cross-Reactive Plasmodium falciparum PfEMP1 Monoclonal Antibody Inhibitory of ICAM-1 Binding.

Lennartz F, Bengtsson A, Olsen RW, Joergensen L, Brown A, Remy L, Man P, Forest E, Barfod LK, Adams Y, Higgins MK, Jensen AT - J. Immunol. (2015)

24E9 mAb recognizes a conformation epitope on PFD1235w DBLβ3_D4. (A) Western blotting of PFD1235w DBLβ3_D4 (D4) and DBLβ3_D5 (D5). +DTT (reduced), −DTT (nonreduced). Lane 1, Prosieve protein marker (M) visualized by phosphorescent paint as dots. Lanes 2 and 3, DBLβ_D4 (±DTT). Lanes 4 and 5, DBLβ3_D5 (±DTT). Arrow shows nonreduced DBLβ3_D4 (lane 3) recognized by 24E9 mAb. (B) 24E9 mAb ELISA reactivity against reduced (+DTT) and nonreduced (−DTT) PFD1235w DBLβ3_D4. (C) AB01 mAb ELISA reactivity against reduced (+DTT) and nonreduced (−DTT) DBLγ of PFD1235w. Mean OD values are shown for three independent experiments. Error bars indicate SD.
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Related In: Results  -  Collection

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fig05: 24E9 mAb recognizes a conformation epitope on PFD1235w DBLβ3_D4. (A) Western blotting of PFD1235w DBLβ3_D4 (D4) and DBLβ3_D5 (D5). +DTT (reduced), −DTT (nonreduced). Lane 1, Prosieve protein marker (M) visualized by phosphorescent paint as dots. Lanes 2 and 3, DBLβ_D4 (±DTT). Lanes 4 and 5, DBLβ3_D5 (±DTT). Arrow shows nonreduced DBLβ3_D4 (lane 3) recognized by 24E9 mAb. (B) 24E9 mAb ELISA reactivity against reduced (+DTT) and nonreduced (−DTT) PFD1235w DBLβ3_D4. (C) AB01 mAb ELISA reactivity against reduced (+DTT) and nonreduced (−DTT) DBLγ of PFD1235w. Mean OD values are shown for three independent experiments. Error bars indicate SD.
Mentions: To determine whether 24E9 mAb interacts with a conformational epitope, we used Western blotting to test the reactivity of 24E9 mAb to reduced and nonreduced DBLβ3_D4. As a control, we performed the same experiment with the non-DC4 PFD1235w DBLβ3_D5 domain. 24E9 recognized only nonreduced DBLβ3_D4 (Fig. 5A). We observed the same result by ELISA (Fig. 5B), where 24E9 recognized only nonreduced DBLβ3_D4. To test whether the loss of reactivity of the mAb toward DTT-treated PFD1235W DBLβ3_D4 was a result of the mAb being reduced in the ELISA, we performed the same assay using the PFD1235w DBLγ-specific human mAb (AB01), which is only partially dependent on the correct folding of DBLγ (24). AB01 mAb was still able to recognize DTT-treated DBLγ (Fig. 5C) showing that a similar mAb remained intact in the ELISA. This suggests that 24E9 mAb targets a conformational epitope.

Bottom Line: When mapped onto a homology model of DBLβ3_D4, these cluster to a defined, surface-exposed region on the convex surface of DBLβ3_D4.Mutagenesis confirmed that the site most strongly protected is necessary for 24E9 binding, which is consistent with a low-resolution structure of the DBLβ3_D4::24E9 Fab complex derived from small-angle x-ray scattering.The convex surface of DBLβ3_D4 has previously been shown to contain the ICAM-1 binding site of DBLβ domains, suggesting that the mAb acts by occluding the ICAM-1 binding surface.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom;

No MeSH data available.


Related in: MedlinePlus