PMID- 7685756
OWN - NLM
STAT- MEDLINE
DCOM- 19930722
LR  - 20210212
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 268
IP  - 18
DP  - 1993 Jun 25
TI  - Purification and characterization of human recombinant IgE-Fc fragments that bind 
      to the human high affinity IgE receptor.
PG  - 13118-27
AB  - The Fc-region of immunoglobulin E (IgE) comprising C epsilon 2, C epsilon 3, and 
      C epsilon 4 domains is sufficient for binding to the alpha chain of the high 
      affinity IgE-Fc receptor (Fc epsilon RI alpha). In order to identify the smallest 
      Fc fragment capable of binding to the Fc epsilon RI alpha with high affinity, 
      various regions of the IgE-Fc molecule were expressed in COS cells and 
      investigated for their ability to bind Fc epsilon RI alpha. The smallest fragment 
      that showed Fc epsilon RI alpha binding activity spans amino acids 329-547 and 
      lacks the entire C epsilon 2 domain. Two active fragments, viz. Fc 
      epsilon(315-547) (containing Cys328 which is responsible for interchain S-S 
      bonding) and Fc epsilon(329-547), have been overexpressed in CHO cells and 
      purified to homogeneity. The purified proteins bind to the Fc epsilon RI alpha 
      with high affinity, similar to native IgE. SDS-polyacrylamide gel electrophoresis 
      analyses indicate that Fc epsilon(315-547) is an S-S-linked dimer of apparent 
      molecular mass of 68 kDa. Fc epsilon(329-547) appears on SDS-gel as three 
      distinct bands at approximately 32 kDa, both under reducing and nonreducing 
      conditions. However, size exclusion chromatography and analytical 
      ultracentrifugation studies suggest that Fc epsilon(329-547) also remains 
      associated as a dimer. The presence of N-linked glycosylation was detected in 
      both proteins. The deglycosylated form of Fc epsilon(315-547) was isolated after 
      Endo F/N-glycosidase F digestion and demonstrated to have binding activity 
      comparable to that of the mock-digested protein. These results suggest that the 
      presence of N-linked sugars is not necessary for Fc epsilon RI alpha binding. 
      Both proteins blocked the release of histamine from RBL cells expressing human Fc 
      epsilon RI alpha in a dose-dependent manner. The availability of these 
      recombinant IgE-Fc proteins will be helpful in elucidating the key epitopes 
      essential for the binding of IgE to its high affinity receptor.
FAU - Basu, M
AU  - Basu M
AD  - Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche 
      Inc., Nutley, New Jersey 07110.
FAU - Hakimi, J
AU  - Hakimi J
FAU - Dharm, E
AU  - Dharm E
FAU - Kondas, J A
AU  - Kondas JA
FAU - Tsien, W H
AU  - Tsien WH
FAU - Pilson, R S
AU  - Pilson RS
FAU - Lin, P
AU  - Lin P
FAU - Gilfillan, A
AU  - Gilfillan A
FAU - Haring, P
AU  - Haring P
FAU - Braswell, E H
AU  - Braswell EH
AU  - et al.
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Immunoglobulin Fc Fragments)
RN  - 0 (Receptors, IgE)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 37341-29-0 (Immunoglobulin E)
SB  - IM
MH  - Animals
MH  - Binding Sites
MH  - Cells, Cultured
MH  - Chromatography, Gel
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Glycosylation
MH  - Histamine Release
MH  - Humans
MH  - Immunoglobulin E/*metabolism
MH  - Immunoglobulin Fc Fragments/isolation & purification/*metabolism
MH  - Rats
MH  - Receptors, IgE/isolation & purification/*metabolism
MH  - Recombinant Fusion Proteins/isolation & purification/metabolism
EDAT- 1993/06/25 00:00
MHDA- 1993/06/25 00:01
CRDT- 1993/06/25 00:00
PHST- 1993/06/25 00:00 [pubmed]
PHST- 1993/06/25 00:01 [medline]
PHST- 1993/06/25 00:00 [entrez]
AID - S0021-9258(19)38627-2 [pii]
PST - ppublish
SO  - J Biol Chem. 1993 Jun 25;268(18):13118-27.