ASSIGNMENT OF DISULFIDE BRIDGES IN BOVINE CD36

The multifunctional membrane protein CD36 is expressed on platelets, m ature monocytes and macrophages, microvascular endothelial cells and m ammary epithelial cells. The exact physiological function of this glyc oprotein is unclear Ln order to determine the number and pattern of di sulfide bridges, CD36 was purified from bovine milk fat globule membra nes. The purification procedure involved Triton X-114 extraction, DEAE -Sepharose ion-exchange chromatography and reverse-phase chromatograph y on a Resource RPC column. The CD36 preparation was used for characte rization of the disulfide bridge pattern, which was determined by pept ide mapping, amino acid sequence analysis, and matrix-assisted laser-d esorption ionization/time of flight mass spectrometry. We have found t hat there are no free cysteines in CD36 and that the six centrally clu stered cysteines are linked by disulfide bonds, Cys242 - Cys310, Cys27 1 - Cys332 and Cys312 - Cys321, resulting in a 1-3, 2-6 and 4-5 arrang ement oi,he disulfide bridges. These data are in agreement with a mode l where the protein is oriented so that it has two short intracellular segments (residues 1-6 and 461-471) and two transmembrane domains (re sidues 7-28 and 439-460), and with four cysteines expected to be acyla ted placed near the intracellular side of the membrane. The remaining part of CD36 is extracellular, comprising eight glycosylations and thr ee disulfide bridges. In the CD36 family of membrane proteins, it is l ikely that a similar pattern of disulfide bridges can be found in the sensory neuron membrane protein-1 from the silk moth Antheraea polyphe mus and the mammalian scavenger receptor class B type I, whereas lysos ome membrane protein II, and epithelial membrane protein from Drosophi la melanogaster are both lacking one cysteine in the area of interest.