Fukutin-related protein resides in the Golgi cisternae of skeletal muscle fibres and forms disulfide-linked homodimers via an N-terminal interaction.

Limb-Girdle Muscular Dystrophy type 2I (LGMD2I) is an inheritable autosomal, recessive disorder caused by mutations in the FuKutin-Related Protein (FKRP) gene (FKRP) located on chromosome 19 (19q13.3). Mutations in FKRP are also associated with Congenital Muscular Dystrophy (MDC1C), Walker-Warburg Syndrome (WWS) and Muscle Eye Brain disease (MEB). These four disorders share in common an incomplete/aberrant O-glycosylation of the membrane/extracellular matrix (ECM) protein α-dystroglycan. However, further knowledge on the FKRP structure and biological function is lacking, and its intracellular location is controversial. Based on immunogold electron microscopy of human skeletal muscle sections we demonstrate that FKRP co-localises with the middle-to-trans-Golgi marker MG160, between the myofibrils in human rectus femoris muscle fibres. Chemical cross-linking experiments followed by pairwise yeast 2-hybrid experiments, and co-immune precipitation, demonstrate that FKRP can exist as homodimers as well as in large multimeric protein complexes when expressed in cell culture. The FKRP homodimer is kept together by a disulfide bridge provided by the most N-terminal cysteine, Cys6. FKRP contains N-glycan of high mannose and/or hybrid type; however, FKRP N-glycosylation is not required for FKRP homodimer or multimer formation. We propose a model for FKRP which is consistent with that of a Golgi resident type II transmembrane protein.

Molecular and cellular characterization of novel {alpha}-mannosidosis mutations.

α-Mannosidosis is a lysosomal storage disorder caused by mutations in the MAN2B1 gene. The clinical presentation of α-mannosidosis is variable, but typically includes mental retardation, skeletal abnormalities and immune deficiency. In order to understand the molecular aetiology of α-mannosidosis, we describe here the subcellular localization and intracellular processing of 35 MAN2B1 variants, including 29 novel missense mutations. In addition, we have analysed the impact of the individual mutations on the three-dimensional structure of the human MAN2B1. We categorize the MAN2B1 missense mutations into four different groups based on their intracellular processing, transport and secretion in cell culture. Impaired transport to the lysosomes is a frequent cause of pathogenicity and correlates with a lack of protein processing (groups 1 and 3). Mutant MAN2B1 proteins that find their way to the lysosomes are processed, but less efficiently than the wild-types (groups 2 and 4). The described four categories of missense mutations likely represent different pathogenic mechanisms. We demonstrate that the severity of individual mutations cannot be determined based only on their position in the sequence. Pathogenic mutations cluster into amino acids which have an important role on the domain interface (arginines) or on the folding of the enzyme (prolines, glycines, cysteines). Tolerated mutations generally include surface mutations and changes without drastic alteration of residue volume. The expression system and structural details presented here provide opportunities for the development of pharmacological therapy by screening or design of small molecules that might assist MAN2B1 folding and hence, transport and activity.