Biochemical and cellular consequences of the antithrombin p.Met1? mutation identified in a severe thrombophilic family

Abstract

Nature is always the best inspiration for basic research. A family with severe thrombosis and antithrombin deficiency, the strongest anticoagulant, carried a new mutation affecting the translation-start codon of SERPINC1, the gene encoding antithrombin. Expression of this variant in a eukaryotic cell system produced three different antithrombins. Two downstream methionines were used as alternative initiation codons, generating highly expressed small aglycosylated antithrombins with cytoplasmic localization. Wild-type antithrombin was generated by the use of the mutated AUU as initiation codon. Actually, any codon except for the three stop codons might be used to initiate translation in this strong Kozak context. We show unexpected consequences of natural mutations affecting translation-start codons. Downstream alternative initiation AUG codons may be used when the start codon is mutated, generating smaller molecules with potential different cell localization, biochemical features and unexplored consequences. Additionally, our data further support the use of other codons apart from AUG for initiation of translation in eukaryotes.

Keywords: antithrombin; initiation codon; thrombosis; translation.

Figure 1. Pedigree of the Norwegian thrombophilic family

The presence of thrombotic events and/or antithrombin deficiency is indicated by symbols filled in black and red, respectively. The arrow points the proband. Carriers who died from thromboembolic events are indicated by ^*. ND: Not determined.

Figure 2. Antithrombins expressed by HEK-EBNA cells 24 h after transfection with pCEP4-S169A (WT) and pCEP4-S169A-M1I (Mut) plasmids

Proteins were identified by SDS-PAGE and western blot using an anti-human antithrombin polyclonal antibody. (A) Antithrombin (AT) released to the conditioned media. The same volume of conditioned was used for cells transfected with both plasmids. (B) Intracellular antithrombin and beta-actin expression. The same amount of cell lysates was used for cells transfected with both plasmids. (C) Glycomic analysis of recombinant antithrombins purified from the conditioned media. The wild-type protein and the small antithrombins were treated (+) or not (-) with N-glycosidase F (PNGase-F) and detected by western blot. Black arrows point the wild-type antithrombin. Grey dashed arrows indicate the small variants. Estimated molecular weights are also indicated.

Figure 3. Anti-thrombin activity of antithrombin from conditioned media of HEK-EBNA cells transfected with wild-type (WT), the pCEP4-S169A-M1I (Mut), pCEP4-S169A-I1X (TAA) and pCEP4-S169A-I1X (TAG) plasmids

The medium was incubated with (+) or without (-) 50 units of thrombin (T_50U). Electrophoretic separation with SDS-PAGE under reducing conditions was followed by immunodetection of antithrombins with an anti-human antithrombin polyclonal antibody. Arrows point native wild-type antithrombin (58 KDa) (N-AT), Thrombin-antithrombin complexes (TAT) (90 KDa), small antithrombins (46 KDa) (S-AT), and cleaved small antithrombins (45 KDa) (cS-AT). Five-fold conditioned media of mutants was loaded compared to the wild-type.

Figure 4. Intracellular expression pattern of antithrombin in HEK-EBNA cells 24 h after transfection with wild-type (WT) and mutant plasmids (Mut)

Wild-type antithrombin is specifically located in the endoplasmic reticulum (ER) and the Golgi complex (G) whereas the mutant is not present in these compartments, showing a cytosolic pattern ER: Endoplasmic reticulum, N: Nucleus, G: Golgi complex, M: Mitochondria. Bars, 200 nm.

Figure 5. Identification of alternative AUG initiation codons in SERPINC1 responsible for the small antithrombins expressed in cells transfected with pCEP4-S169A-M1I (Mut) plasmid

(A) Squematic representation of AUG codons identified in the pCEP4-S169A (WT) and pCEP4-S169A-M1I (Mut) plasmids used for transfection. Potential initiation codons, localization of the signal peptide, and the mature proteins with potential post-translational modifications are shown. S: disulfide linked bonds; ^* N-glycosylation. (B) Antithrombin (AT) released to conditioned media of cells transfected with WT and Mut plasmids, as well as with mutated pCEP4-S169A-M1I plasmids that also contained nonsense mutations for Met49 and Met52. Antithrombins were immunodetected after Western blot. Full length wild-type antithrombin (58 KDa) is pointed by an arrow while small antithrombins (46 KDa) are pointed by grey arrows. Five-fold conditioned media of mutants was loaded compared to the wild-type.

Figure 6. Identification of non AUG initiation codons in SERPINC1

(A) Squematic representation of the mutated pCEP4-S169A-M1I plasmid used in the recombinant model showing residues mutated to identify alternative initiation codons. Triangles represent stop codons in the reading frame of SERPINC1. (B) Antithrombin released to conditioned media (AT) of cells transfected with the pCEP4-S169A-M1I plasmid containing different mutations: missense or nonsense mutations affecting residues located before the original initiation codon (L-5V, L-5X; G-4X; T-1X); missense or nonsense mutations at or just after the original initiation codon (I1S, I1X, N4X); nonsense mutation affecting the first downstream AUG codons (M49X, M52X); and a nonsense mutation downstream of all potential initiation codons (R161X). (C) Intracellular expression of beta-actin as loading control for selected mutants, including those with no antithrombin released to the conditioned media (M52X and R161X). (D) Consequence on antithrombin from conditioned media of mutations affecting Ile1 in the pCEP4-S169A-M1I plasmid. (E) Effect on antithrombin from conditioned media of possible nonsense mutations affecting position 1 in the pCEP4-S169A-M1I plasmid. (F) Squematic representation of the wild-type pCEP4-S169A plasmid showing the mutation that severely disturbs the Kozak sequence (T-1X) in a wild-type context (Met1). Antithrombin from conditioned media and cell lysate as well as intracellular beta-actin were detected by Western blot. Full length wild-type antithrombin (58 KDa) is pointed by an arrow while small antithrombins (46 KDa) are pointed by grey arrows. Five-fold conditioned media of mutants was loaded compared to the wild-type.