INTRACELLULAR RETENTION OF A FACTOR-VIII PROTEIN WITH AN ARG(2307)-]GLN MUTATION AS A CAUSE OF HEMOPHILIA-A

Substitution of Arg(2307) by Gin in factor VIII has been found to be a ssociated with mild to moderate haemophilia A [Gitschier, Wood, Shuman and Lawn (1986) Science 232, 1415-1416]. We have introduced this part icular point mutation into a B-domain-deleted factor VIII cDNA and exp ressed the modified cDNA in C127 cells. Cells expressing the resulting protein, termed des-(868-1562)-factor VIII-R2307Q, were compared with those expressing the previously characterized des-(868-1562)-factor V III. No immunoreactive material could be detected in the conditioned m edium of cells transfected with des-(868-1562)-factor VIII-R2307Q cDNA using assays specific for the factor VIII light chain and the factor VIII heavy chain. Analysis of metabolically labelled cells transfected with des-(868-1562)-factor VIII-R2307Q cDNA revealed that this mutant protein is synthesized at a level similar to des-(868-1562)-factor VI II. In contrast to des-(868-1562)-factor VIII, metabolically labelled des-(868-1562)-factor VIII-R2307Q was not encountered in the condition ed medium of the transfected cells, indicating that the mutant protein is not secreted from the cell. Inspection of the intracellular locali zation of the two proteins in the cell employing morphological analysi s, endoglycosidase H and experiments with inhibitors of glucosidases I and II was consistent with focalization of des-(868-1562)-factor VIII and des-(868-1562)-factor VIII-R2307Q in the endoplasmic reticulum. T aken together, our data indicate that the Arg(2307) --> Gln mutation r esults in aberrant intracellular trafficking of factor VIII, which may explain the low levels of factor WI antigen in the plasma of haemophi lia A patients that carry this particular point mutation.