SUMMARY Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192→Met) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on α-dystroglycan that is required for high-affinity binding to laminin.

The interaction between epithelial cells and the extracellular matrix is crucial for tissue architecture and function and is compromised during cancer progression. Dystroglycan is a membrane receptor that mediates interactions between cells and basement membranes in various epithelia. In many epithelium-derived cancers, ␤-dystroglycan is expressed, but ␣-dystroglycan is not detected. Here we report that ␣-dystroglycan is correctly expressed and trafficked to the cell membrane but lacks laminin binding as a result of the silencing of the like-acetylglucosaminyltransferase (LARGE) gene in a cohort of highly metastatic epithelial cell lines derived from breast, cervical, and lung cancers. Exogenous expression of LARGE in these cancer cells restores the normal glycosylation and laminin binding of ␣-dystroglycan, leading to enhanced cell adhesion and reduced cell migration in vitro. Our findings demonstrate that LARGE repression is responsible for the defects in dystroglycan-mediated cell adhesion that are observed in epithelium-derived cancer cells and point to a defect of dystroglycan glycosylation as a factor in cancer progression.Normal epithelial cells are tightly associated with one another and with the underlying basement membrane to maintain tissue architecture and function. During cancer progression, primitive cancer cells escape from this control by modifying the binding affinities of their cell membrane receptors. Several receptors have been described as important for this process. Of these, the integrins are the best studied (1). The receptor dystroglycan has been reported to be required for the development and maintenance of epithelial tissues (2, 3). A direct requirement for dystroglycan in epithelia is further demonstrated by the profound effect that loss of dystroglycan expression has on cell polarity and laminin binding in cultured mammary epithelial cells (4, 5). However, dystroglycan is not only important in the establishment and maintenance of epithelial structure. Associations have also been made between the loss of ␣-dystroglycan immunoreactivity and cancer progression in tumors of epithelial origin, including breast, colon, cervix, and prostate cancers (4, 6 -9). The dystroglycan loss of function could thus serve as an effective means by which cancerous cells modify their adhesion to the extracellular matrix (ECM). 2Dystroglycan is a ubiquitously expressed cell membrane protein that plays a key function in cellular integrity, linking the intracellular cytoskeleton to the extracellular matrix. The dystroglycan gene encodes a preprotein that is cleaved into two peptides (10). The C-terminal component, known as ␤-dystroglycan, is embedded within the cell membrane, whereas the N-terminal component, ␣-dystroglycan, is present within the extracellular periphery but remains associated with ␤-dystroglycan through non-covalent bonds. ␤-Dystroglycan binds to actin (11), dystrophin (11), utrophin (11), and Grb2 (12) through its C-terminal intracellular domain. ␣-Dystroglycan, on the other hand, binds to E...

Background: Dystroglycan function is decreased in prostate and other cancers. Results: Decreased functional glycosylation of ␣-dystroglycan is clinically correlated with reduced LARGE2 and restoring its expression rescues dystroglycan function, reducing the invasive and proliferative potential of prostate cancer cells. Conclusion: LARGE2 regulates ␣-dystroglycan function in the prostatic epithelium. Significance: Loss of LARGE2 expression may contribute to prostate cancer progression.

Intragenic homozygous deletions in the Large gene are associated with a severe neuromuscular phenotype in the myodystrophy (myd) mouse. These mutations result in a virtual lack of glycosylation of -dystroglycan. Compound heterozygous LARGE mutations have been reported in a single human patient, manifesting with mild congenital muscular dystrophy (CMD) and severe mental retardation. These mutations are likely to retain some residual LARGE glycosyltransferase activity as indicated by residual -dystroglycan glycosylation in patient cells. We hypothesized that more severe LARGE mutations are associated with a more severe CMD phenotype in humans. Here we report a 63-kb intragenic LARGE deletion in a family with WalkerWarburg syndrome (WWS), which is characterized by CMD, and severe structural brain and eye malformations. This Wnding demonstrates that LARGE gene mutations can give rise to a wide clinical spectrum, similar as for other genes that have a role in the post-translational modiWcation of the -dystroglycan protein.

Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles.

Alkaptonuria (AKU), the prototypic inborn error of metabolism, was the first human disease to be interpreted as a Mendelian trait by Garrod and Bateson at the beginning of last century. AKU results from impaired function of homogentisate dioxygenase (HGO), an enzyme required for the catabolism of phenylalanine and tyrosine. With the novel 7 AKU and 22 fungal mutations reported here, a total of 84 mutations impairing this enzyme have been found in the HGO gene from humans and model organisms. Forty-three of these mutations result in single amino acid substitutions. This mutational information is analysed here in the context of the HGO structure and function using kinetic assays performed using purified AKU mutant enzymes and the crystal structure of human HGO. HGO is a topologically complex structure which assembles as a functional hexamer arranged as a dimer of trimers. We show how the intricate pattern of intra- and inter-subunit interactions and the extensive surfaces required for subunit folding and association of this oligomeric enzyme can be inactivated at multiple levels by single-residue substitutions. This explains, in part, the predominance of missense mutations (67%) in AKU.

Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometrybased proteomics methods.

Alkaptonuria (AKU; McKusick No. 203500), a rare hereditary disorder of the phenylalanine catabolism, was the first disease to be interpreted as an inborn error of metabolism (A. E. Garrod, 1902, Lancet 2: 1616-1620). AKU patients are deficient for homogentisate 1,2-dioxygenase (HGO; EC 1.13.11.5). This enzymatic deficiency causes homogentisic aciduria, ochronosis, and arthritis. Recently we cloned the human HGO gene and showed that AKU patients carry two copies of a loss-of-function HGO allele. Here we describe the complete nucleotide sequence of the human HGO gene and the identification of its promoter region. The human HGO gene spans 54,363 bp and codes for a 1715-nt-long transcript that is split into 14 exons ranging from 35 to 360 bp. The HGO introns, 605 to 17,687 bp in length, contain representatives of the major classes of repetitive elements, including several simple sequence repeats (SSR). Two of these SSRs, a (CT)n repeat in intron 4 and a (CA)n repeat in intron 13, were found to be polymorphic in a Spanish population sample. The HGO transcription start site was determined by primer extension. We report that sequences from -1074 to +89 bp (relative to the HGO transcription start site) are sufficient to promote transcription of a CAT reporter gene in human liver cells and that this fragment contains putative binding sites for liver-enriched transcription factors that might be involved in the regulation of HGO expression in liver.