Purification and molecular characterization of FAP, a feto-acinar protein associated with the differentiation of human pancreas.
This work describes the purification of FAP, a feto-acinar pancreatic protein associated with the ontogenesis, differentiation, and transformation of the human exocrine pancreas. The protein was purified to homogeneity from pancreatic juices of patients with pancreatic pathology by a two-step chromatographic procedure which consisted of size exclusion on Sephacryl S-200 and affinity on heparin-Sepharose. The final preparation gave a single band at Mr 110,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after Coomassie stain or autoradiography of the 125I-labeled protein. Immunodetection with the murine monoclonal antibody mAb J28 in nitrocellulose replicas demonstrated a main Mr 110,000 component and trace components in the Mr 100,000-80,000 range. The immunopattern was identical to that in the original crude pancreatic secretion, therefore showing that the molecular characteristics of the protein, i.e. molecular mass, microheterogeneity, and immunoreactivity, were not altered along the purification procedure. FAP was identified as an acidic protein (isoelectric point 4.2-4.8) consisting of a single polypeptide chain having no free SH residues. Analysis of the amino acid composition showed a high proline content. Twenty-two residues of the N-terminal sequence were determined. No significant homology between this peptide and other proteins was found following a search of the NBRF-18 data bank. Sugar analysis showed the presence of mannose which is consistent with N-linked carbohydrate chains and an unusual high ratio in N-acetylgalactosamine residues suggesting the presence of many O-linked carbohydrate chains. Sequential deglycosylation with neuraminidase, hexosaminidase, and O-glycanase yield a single Mr 58,000 peptide showing that, relative to a molecular mass of 110,000, the carbohydrate moiety of FAP accounts for at least 47% of its apparent Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Escribano
M J
MJ
Laboratoire d'Immunochimie, UPR 277 du Centre National de la Recherche Scientifique, Villejuif, France.
Imperial
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000502:physiology; D002454:Cell Differentiation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008049:Lipase; D008969:Molecular Sequence Data; D010179:Pancreas / Q000166:cytology*; D010189:Pancreatic Juice / Q000032:analysis*
D000596:Amino Acids; D002241:Carbohydrates; D002352:Carrier Proteins; D006023:Glycoproteins; C054621:CEL protein, human; D008049:Lipase