Impaired Cotranslational Processing as a Mechanism for Type I Antithrombin Deficiency

Most secretory proteins, including antithrombin (AT), aresynthesized with a signal peptide, which is cleaved before the mature protein is exported from the cell. The signal peptide is important inthe process whereby nascent protein is recognized as requiring subsequent modification within the endoplasmic reticulum (ER). We haveidentified a novel mutation, 2436TC L(-10)P, which affects the central hydrophobic domain of the AT signal peptide, in a probandpresenting with venous thrombotic disease and type I AT deficiency. Weinvestigated the basis of the phenotype by examining expression inmammalian cells of a range of variant AT cDNAs with mutations affectingthe -10 residue. Glycosylated AT was secreted from COS-7 cellstransfected with wild-type AT, -10L deletion, -10V or -10M variants,but not variants with P, T, R, or G at -10. Cell-free expression ofwild-type and variant AT cDNAs was then performed in the presence ofcanine pancreatic microsomes, as a substitute for ER. Variant ATproteins with P, T, R, or G at residue -10 did not undergoposttranslational glycosylation, and their susceptibility to trypsindigestion suggested they had not been translocated into microsomes. Ourresults suggest that the ability of AT signal peptide to direct theprotein to ER for cotranslational processing events appears to becritically dependent on maintaining the hydrophobic nature of theregion including residue -10. The investigations have defined impairedcotranslational processing as a hitherto unrecognized cause ofhereditary AT deficiency.