Structure-function analysis of decay-accelerating factor: Identification of residues important for binding of the Escherichia coli Dr adhesin and complement regulation

Rafia J. Hasan, Edyta Pawelczyk, Petri T. Urvil, Mathura S. Venkatarajan, Pawel Goluszko, Jozef Kur, Rangaraj Selvarangan, Stella Nowicki, Werner Braun, Bogdan J. Nowicki

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesin-bearing Escherichia coli. The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation. However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a-); these cells contain a point mutation (Ser165-Leu) in DAF repeat three. In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF. Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids - predominantly those in close proximity to Ser165 to alanine - and expressed these mutations in Chinese hamster ovary cells. To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs. We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin. The IH4 binding epitope contains residues Phe148, Ser155, and L171. Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation. Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are ≈20 Å apart.

Original languageEnglish (US)
Pages (from-to)4485-4493
Number of pages9
JournalInfection and Immunity
Volume70
Issue number8
DOIs
StatePublished - 2002
Externally publishedYes

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Escherichia coli Adhesins
CD55 Antigens
Amino Acids
Epitopes
Complement System Proteins
Poxviridae
Mutation
Cricetulus
Point Mutation
Alanine
Ovary
Erythrocytes
Monoclonal Antibodies

ASJC Scopus subject areas

  • Immunology

Cite this

Structure-function analysis of decay-accelerating factor : Identification of residues important for binding of the Escherichia coli Dr adhesin and complement regulation. / Hasan, Rafia J.; Pawelczyk, Edyta; Urvil, Petri T.; Venkatarajan, Mathura S.; Goluszko, Pawel; Kur, Jozef; Selvarangan, Rangaraj; Nowicki, Stella; Braun, Werner; Nowicki, Bogdan J.

In: Infection and Immunity, Vol. 70, No. 8, 2002, p. 4485-4493.

Research output: Contribution to journalArticle

Hasan, Rafia J. ; Pawelczyk, Edyta ; Urvil, Petri T. ; Venkatarajan, Mathura S. ; Goluszko, Pawel ; Kur, Jozef ; Selvarangan, Rangaraj ; Nowicki, Stella ; Braun, Werner ; Nowicki, Bogdan J. / Structure-function analysis of decay-accelerating factor : Identification of residues important for binding of the Escherichia coli Dr adhesin and complement regulation. In: Infection and Immunity. 2002 ; Vol. 70, No. 8. pp. 4485-4493.
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AU - Hasan, Rafia J.

AU - Pawelczyk, Edyta

AU - Urvil, Petri T.

AU - Venkatarajan, Mathura S.

AU - Goluszko, Pawel

AU - Kur, Jozef

AU - Selvarangan, Rangaraj

AU - Nowicki, Stella

AU - Braun, Werner

AU - Nowicki, Bogdan J.

PY - 2002

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N2 - Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesin-bearing Escherichia coli. The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation. However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a-); these cells contain a point mutation (Ser165-Leu) in DAF repeat three. In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF. Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids - predominantly those in close proximity to Ser165 to alanine - and expressed these mutations in Chinese hamster ovary cells. To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs. We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin. The IH4 binding epitope contains residues Phe148, Ser155, and L171. Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation. Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are ≈20 Å apart.

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