TY - JOUR
T1 - Structure-function analysis of decay-accelerating factor
T2 - Identification of residues important for binding of the Escherichia coli Dr adhesin and complement regulation
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 A.
AU - Nowicki, Bogdan J.
PY - 2002
Y1 - 2002
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.
AB - 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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U2 - 10.1128/IAI.70.8.4485-4493.2002
DO - 10.1128/IAI.70.8.4485-4493.2002
M3 - Article
C2 - 12117960
AN - SCOPUS:0036073625
SN - 0019-9567
VL - 70
SP - 4485
EP - 4493
JO - Infection and immunity
JF - Infection and immunity
IS - 8
ER -