Purification and characterization of a 95-kDa protein--carcinoembryonic antigen-related cell adhesion molecule 8--from normal human granulocytes.
A 95-kDa protein was purified to homogeneity from granule extracts of normal human granulocytes. The column procedure consisted of Sephadex G-75, Mono-S cation exchange and Superdex HR 75 chromatography. The purified protein showed only one broad band at a molecular weight of 95 kDa when analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It reacted with polyclonal antibodies against carcinoembryonic antigen (CEA) and a specific monoclonal antibody against CD66b, but did not react with monoclonal antibodies against CD66acde and CD66c when analyzed by immunoblotting. The molecular weight of the protein shifted from 95 to 40 kDa on SDS-PAGE after deglycosylation. Tryptic peptide analysis by MALDI-Tof identified four peptides with spectra of m/z matching the expected tryptic peptides from a CGM6 gene product. Furthermore, the nanoelectrospray mass spectrometry (MS/MS) analysis of the two selected tryptic peptides of the protein revealed two amino acid sequences corresponding to residues 79-98 and 199-207 of the CGM6 gene product. Based on this, and also on the immunochemical data, it is concluded that the purified 95 kDa is identical to carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8) (nonspecific cross-reacting antigen (NCA)-95, CD67 and CD66b) and is a product of the CGM6 (W272) gene. We present, for the first time, a method for the purification of CEACAM8 from normal human granulocytes, which should be useful for further studies on its structure and functions. We also confirmed at the protein level that CEACAM8 is a product of the CGM6 (NCA-W272) gene.
Zhao
Linshu
L
Department of Medical Sciences, Clinical Chemistry, Uppsala University, SE-751 85 Uppsala, Sweden.
Xu
Shengyuan
S
Peterson
Christer
C
Kuroki
Motomu
M
Kuroki
Masahide
M
Venge
Per
P
D000596:Amino Acids / Q000032:analysis; D015703:Antigens, CD; D000951:Antigens, Neoplasm / Q000032:analysis*; D015815:Cell Adhesion Molecules / Q000032:analysis*; D058851:GPI-Linked Proteins; D006098:Granulocytes / Q000737:chemistry*; D006801:Humans; D010455:Peptides / Q000032:analysis
D000596:Amino Acids; D015703:Antigens, CD; D000951:Antigens, Neoplasm; C504117:CEACAM6 protein, human; C504120:CEACAM8 protein, human; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D010455:Peptides