Impaired cotranslational processing as a mechanism for type I antithrombindeficiency

Most secretory proteins, including antithrombin (AT), are synthesized with a signal peptide, which is cleaved before the mature protein is exported fr om the cell. The signal peptide is important in the process whereby nascent protein is recognized as requiring subsequent modification within the endo plasmic reticulum (ER). We have identified a novel mutation, 2436T-->C L(-1 0)P, which affects the central hydrophobic domain of the AT signal peptide, in a proband presenting with venous thrombotic disease and type I AT defic iency. We investigated the basis of the phenotype by examining expression i n mammalian cells of a range of variant AT cDNAs with mutations affecting t he -10 residue. Glycosylated AT was secreted from COS-7 cells transfected w ith wild-type AT, -10L deletion, -10V or -10M variants, but not variants wi th P, T, R, or G at -10. Cell-free expression of wild-type and variant AT c DNAs was then performed in the presence of canine pancreatic microsomes, as a substitute for ER. Variant AT proteins with P, T, R, or G at residue -10 did not undergo posttranslational glycosylation, and their susceptibility to trypsin digestion suggested they had not been translocated into microsom es. Our results suggest that the ability of AT signal peptide to direct the protein to ER for cotranslational processing events appears to be critical ly dependent on maintaining the hydrophobic nature of the region including residue -10. The investigations have defined impaired cotranslational proce ssing as a hitherto unrecognized cause of hereditary AT deficiency. (C) 199 8 by The American Society of Hematology.