Expression of wild-type, As135Thr, and Ser137Ala antithrombins. (A) Antithrombin variants were expressed using rabbit reticulocyte lysate and coupled to posttranslational modification by the addition of microsomal membranes (+). Products above 52 kD in the coupled reactions represent N-linked glycosylation products. The uppermost of the doublet glycosylation isoform (*) produced from the wild-type protein (Wt) is absent in two variants with substitutions affecting the N-linked consensus glycosylation site at residue 135 (N135T and S137A, labelled 135 and 137, respectively); see lanes 1 through 3, but more clearly in lanes 4 through 6, where trypsin has been added (+) after protein has been incubated with microsomes, and protein not translocated into the protective environment of the microsomes has been degraded. Lanes 7 through 9 are control incubations of wild-type and two variant antithrombins with endoglycosidase H (+) and indicate that this enzyme strips the carbohydrate side chains from the high MW forms. The band labelled 47 kD results from internal initiation of translation at Met17. (B) Antithrombin was expressed in COS-7 cells. Supernatant of COS-7 cells transfected with antithrombin constructs was immunoprecipitated using polyclonal antithrombin antibody. Glycosylated antithrombin was found in the supernatants with a size of approximately 60 kD. At least two glycosylation isoforms were observed for wild-type (WT) antithrombin (just above and below the 60-kD arrow), but the larger form was not observed in the N135T or S137A variants. Several nonspecific bands are apparent in each lane and are also seen in supernatants from mock-transfected COS cells (not shown).