As for O-linked glycosylation, the presence of at least seven O-l

As for O-linked glycosylation, the presence of at least seven O-linked carbohydrate structures glycans in the FVIII B-domain has been reported [3,4]. Unfortunately, no information Trichostatin A cost on their location and precise structure is available, except for the detection of a single di-sialylated T-antigen at position Ser750 in a recombinant B-domainless FVIII preparation

[5]. Interestingly, comparison of pd-FVIII and rFVIII has revealed a number of subtle differences in their glycan structures. First, pd-FVIII but not rFVIII seems to carry blood-group determinants on its biantennary sugar chains [1]. Second, Hironaka et al. detected the presence of Gal(α1-3) Gal structures on rFVIII derived from BHK-cells but not on pd-FVIII. The incorporation of Gal(α1-3) Gal structures requires the presence of α1,3-galactosyltransferase, an enzyme that is mainly expressed in cells of non-primate find more origin, the type of cells used for the production of current rFVIII products. Third, human cells differ from other mammalian cells in that they express a non-functional variant of cytidine monophosphate N-acetylneuraminic acid hydroxylase, which catalyses the conversion

of the sialic acid N-acetyl neuraminic acid (Neu5Ac) into N-Glycolylneuraminic acid (Neu5Gc) [6]. It is possible therefore that rFVIII contains both Neu5Ac and Neu5Gc, whereas pd-FVIII selectively carries the Neu5Ac sialic acid. Of note, the non-human nature of Gal(α1-3)Gal and Neu5Gc structures has been associated with the development of antibodies against these structures, and the presence of, for instance, the Gal(α1-3)Gal moiety on biotherapeutics can cause adverse events [6,7]. Whether these structures contribute to the immune response following treatment with rFVIII is unknown, but so far no evidence in support of this possibility has been provided. Intracellular

processing and routing of FVIII requires the interaction of this molecule with a number of glycan-binding chaperones, including calnexin, calreticulin and the LMAN1(ERGIC53)/MCFD2 complex (for a more detailed review see [8]). Calnexin and calreticulin are resident ER chaperone proteins that interact with mono-glucosylated carbohydrate structures that are present on proteins in the early stage of synthesis, and are part of the quality control system MCE of the cell. The interaction with both chaperones is necessary for optimal protein folding. Once properly folded, trimming of the terminal glucose residue results in dissociation of the protein from both chaperones, allowing the protein to be routed from the ER to the Golgi. With regard to FVIII, it has been shown that the glycans in the B-domain play an important role in the interaction with calreticulin/calnexin [9]. The transition from the ER to the Golgi involves the interaction between FVIII and the LMAN1 (ERGIC-53)/MCFD2 complex.

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