Assignment of disulfide bridges in bovine CD36.
The multifunctional membrane protein CD36 is expressed on platelets, mature monocytes and macrophages, microvascular endothelial cells and mammary epithelial cells. The exact physiological function of this glycoprotein is unclear. In order to determine the number and pattern of disulfide bridges, CD36 was purified from bovine milk fat globule membranes. The purification procedure involved Triton X-114 extraction, DEAE-Sepharose ion-exchange chromatography and reverse-phase chromatography on a Resource RPC column. The CD36 preparation was used for characterization of the disulfide bridge pattern, which was determined by peptide mapping, amino acid sequence analysis, and matrix-assisted laser-desorption ionization/time of flight mass spectrometry. We have found that there are no free cysteines in CD36 and that the six centrally clustered cysteines are linked by disulfide bonds, Cys242-Cys310, Cys271-Cys332 and Cys312-Cys321, resulting in a 1-3, 2-6 and 4-5 arrangement of the disulfide bridges. These data are in agreement with a model where the protein is oriented so that it has two short intracellular segments (residues 1-6 and 461-471) and two transmembrane domains (residues 7-28 and 439-460), and with four cysteines expected to be acylated placed near the intracellular side of the membrane. The remaining part of CD36 is extracellular, comprising eight glycosylations and three disulfide bridges. In the CD36 family of membrane proteins, it is likely that a similar pattern of disulfide bridges can be found in the sensory neuron membrane protein-1 from the silk moth Antheraea polyphemus and the mammalian scavenger receptor class B type I, whereas lysosome membrane protein II, and epithelial membrane protein from Drosophila melanogaster are both lacking one cysteine in the area of interest.
Rasmussen
J T
JT
Protein Chemistry Laboratory, University of Aarhus, Denmark.
Berglund
L
L
Rasmussen
M S
MS
Petersen
T E
TE
D000595:Amino Acid Sequence; D000818:Animals; D018955:CD36 Antigens / Q000737:chemistry* / Q000302:isolation & purification; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008892:Milk / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018955:CD36 Antigens; D004220:Disulfides; D010446:Peptide Fragments