Purification and analysis of an 80-kDa carcinoembryonic antigen-binding protein from Kupffer cells.

The receptor-mediated interaction of Kupffer cells with carcinoembryonic antigen (CEA) has led to the identification of an 80-kDa CEA-binding Kupffer cell protein. This study is aimed at the isolation and analyses of this protein from rat Kupffer cells. The binding protein was purified using a combination of gel filtration, preparative polyacrylamide gel electrophoresis (PPAGE), and affinity chromatography using a CEA-Sepharose column. Fractions obtained from the gel filtration produced two major and few minor peaks with CEA-binding activity. Maximum reactivity was detected in the first major peak. The first major peak protein was partially precipitated following fractionation with 30% loss of activity in the precipitate. Fractions with CEA-binding activity were pooled and separated on the basis of molecular weight (MW) in PPAGE. The fractions between MW 70 and 90 kDa were pooled and affinity purified using CEA-Sepharose affinity chromatography. The purity of the 80-kDa protein was demonstrated by a single protein band on SDS-polyacrylamide gel. The protein was further identified by an anti-80-kDa binding protein antibody in Western blot analysis. The pI of the 80-kDa protein is 4.95. Amino acid analysis demonstrated no histidine; higher percentages of glutamine (13.3%), leucine (11.2%), asparagine and alanine (10.4%), and lysine (9.2%) were observed. Protein microsequencing revealed two unique sequences, one with 16 amino acids and the other with 11 amino acids. The 16-amino-acid sequence has less than 50% homology with a large sample of unrelated proteins, whereas the sequence containing 11 amino acids has 60-70% homology with the alpha chain of collagen from a variety of species but no significant homology with other known proteins, suggesting the presence of collagen-like domains in the 80-kDa receptor.