Mutant glycosyltransferase and altered glycosylation of alpha-dystroglycan in the myodystrophy mouse.

Spontaneous and engineered mouse mutants have facilitated our understanding of the pathogenesis of muscular dystrophy and they provide models for the development of therapeutic approaches. The mouse myodystrophy (myd) mutation produces an autosomal recessive, neuromuscular phenotype. Homozygotes have an abnormal gait, show abnormal posturing when suspended by the tail and are smaller than littermate controls. Serum creatine kinase is elevated and muscle histology is typical of a progressive myopathy with focal areas of acute necrosis and clusters of regenerating fibers. Additional aspects of the phenotype include sensorineural deafness, reduced lifespan and decreased reproductive fitness. The myd mutation maps to mouse chromosome 8 at approximately 33 centimorgans (cM) (refs. 2, 4-7). Here we show that the gene mutated in myd encodes a glycosyltransferase, Large. The human homolog of this gene (LARGE) maps to chromosome 22q. In myd, an intragenic deletion of exons 4-7 causes a frameshift in the resultant mRNA and a premature termination codon before the first of the two catalytic domains. On immunoblots, a monoclonal antibody to alpha-dystroglycan (a component of the dystrophin-associated glycoprotein complex) shows reduced binding in myd, which we attribute to altered glycosylation of this protein. We speculate that abnormal post-translational modification of alpha-dystroglycan may contribute to the myd phenotype.

A novel mechanism for modulating synaptic gene expression: differential localization of alpha-dystrobrevin transcripts in skeletal muscle.

Alpha-dystrobrevin is a dystrophin-related and -associated protein that is involved in synapse maturation and is required for normal muscle function. There are three protein isoforms in skeletal muscle, alpha-dystrobrevin-1, -2, and -3 that are encoded by the single alpha-dystrobrevin gene. To understand the role of these proteins in muscle we have investigated the localisation and transcript distribution of the different alpha-dystrobrevin isoforms. Alpha-dystrobrevin-1 and -2 are concentrated at the neuromuscular junction and are both recruited into agrin-induced acetylcholine receptor clusters in cultured myotubes. We also demonstrate that all alpha-dystrobrevin mRNAs are transcribed from a single promoter in skeletal muscle. However, only transcripts encoding alpha-dystrobrevin-1 are preferentially accumulated at postsynaptic sites. These data suggest that the synaptic accumulation of alpha-dystrobrevin-1 mRNA occurs posttranscriptionally, identifying a novel mechanism for synaptic gene expression. Taken together, these results indicate that different isoforms possess distinct roles in synapse formation and possibly in the pathogenesis of muscular dystrophy.

A second promoter provides an alternative target for therapeutic up-regulation of utrophin in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5' end of the gene and have shown it to be synaptically regulated in vivo. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3' to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.

Tissue-selective expression of alpha-dystrobrevin is determined by multiple promoters.

alpha-Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a dystrophin-associated and dystrophin-related protein. Knockout of the gene in the mouse results in muscular dystrophy. The control of the alpha-dystrobrevin gene in the various tissues is therefore of interest. Multiple dystrobrevin isoforms differing in their domain content are generated by alternative splicing of a single gene. The data presented here demonstrate that expression of alpha-dystrobrevin from three promoters, that are active in a tissue-selective manner, also plays a role in the function of the protein in different tissues. The most proximal promoter A is active in brain and to a lesser extent in lung, whereas the most distal promoter B, which possesses several Sp1 binding sites, is restricted to brain. Promoter C, which contains multiple consensus myogenic binding sites, is up-regulated during in vitro myoblast differentiation. Interestingly, the organization and the activity of the alpha-dystrobrevin promoters is reminiscent of those in the dystrophin gene. Taken together we suggest that the multipromoter system, distributed over a region of 270 kilobases at the 5'-end of the alpha-dystrobrevin gene, has been developed to allow the regulation of this gene in different cell types and/or different developmental stages.

Structure and organization of the porcine LCN1 gene encoding Tear lipocalin/von Ebner's gland protein.

We have cloned and sequenced the entire genomic fragment of the porcine LCN1 gene which encodes Tear lipocalin/von Ebner's gland protein, a member of the lipocalin superfamily highly expressed in porcine lachrymal and lingual glands. The porcine LCN1 gene is approximately 4.6 kb in size and contains six protein-coding exons and a 3'-nontranslated exon. The structure of this porcine gene is highly similar, in terms of numbers of exons/introns, in size of exons and in intron phasing, to that of the human LCN1 and rat VEGP genes, thus supporting a very close evolutionary relationship of these genes. Within the promoter region of the porcine LCN1 a putative TATA box, a CAAT box and two MRE motifs are found. The same MRE motifs are conserved in the human LCN1 promoter, suggesting that they might be of relevance for LCN1 gene expression. However, additional motifs present in the human LCN1 promoter, such as AP-1 and AP-2 sites, a NF-kappaB site, and a cAMP-responsive element, could not be detected in the porcine LCN1 promoter.

Expression of the gene for tear lipocalin/von Ebner's gland protein in human prostate.

Northern analysis of human multiple tissue blots containing poly A+ RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes revealed that a prostate specific transcript hybridizes to a tear lipocalin/von Ebner's gland protein (TL/VEGP) gene probe. To characterize this transcript, the corresponding cDNA was amplified by reverse transcription (RT)-PCR. Cloning and sequence analysis showed that it was identical to the tear lipocalin cDNA isolated from human lachrymal glands. Immunohistochemical analysis on thin layer sections of human prostate using a tear lipocalin specific antiserum confirmed the expression of this cDNA in prostate. Thus, our results clearly argue against a unique function of TL/VEGP in human tear fluid or saliva. The human cDNA was expressed in E. coli using the pQE system yielding a recombinant protein which shows biochemical properties identical to the native TL/VEGP.

The human lacrimal gland synthesizes apolipoprotein D mRNA in addition to tear prealbumin mRNA, both species encoding members of the lipocalin superfamily.

Apolipoprotein D (apoD), a glycoprotein originally characterized as a component of the high density lipoprotein fraction of human plasma and known to be a member of the lipocalin protein superfamily, has been found in human tear fluid by Western blot analysis. Unlike serum it seems that in the tear fluid apoD exists mainly as a disulphide linked homodimer which is not associated with lecithin/cholesterol acyltransferase (LCAT) or apolipoprotein A-I (apo A-I). By reverse-transcription-PCR (RT-PCR) of mRNA extracted from a human lacrimal gland and use of specific primers we could demonstrate expression of the apoD gene in this tissue. The amplified cDNA was cloned and a subsequent sequence analysis confirmed the identity of apoD mRNA in the human lacrimal gland. These investigations indicate that the lacrimal gland is the site of synthesis of the tear fluid apoD. Although the physiological function of apoD is unknown, it has the ability to bind phospholipids, cholesterol and other small hydrophobic molecules. Therefore, this protein might interact with meibomian lipids present in human tear fluid and probably contribute to the surface spreading of these lipids or it may function as a clearance factor, protecting the cornea from harmful lipophilic molecules.

Confirmation of 9q34 as the chromosomal site of the human lipocalin LCN1 gene.

The gene encoding the lipocalin LCN1 was previously assigned to human chromosome 8q24 by fluorescence in situ hybridization of a genomic DNA fragment and to 9q34 by in situ hybridization of a radiolabeled cDNA fragment of LCN1. To resolve this discrepancy, we used hamster x human hybrid cells for polymerase chain reaction experiments. These investigations confirmed the localization of LCN1 on chromosome 9.

Structural organization of the gene encoding the human lipocalin tear prealbumin and synthesis of the recombinant protein in Escherichia coli.

The genomic DNA fragment encoding the human lipocalin tear prealbumin (LCN1), a new member of the superfamily of hydrophobic molecule transporters, has been isolated and sequenced. The entire gene is approximately 6.2 kb in size and contains six protein-coding exons and a 3'-nontranslated exon. All exon/intron splice junctions exactly follow the GT/AG rule. The structure of the LCN1 gene is highly similar, in terms of numbers and sizes of exons and in intron phasing, to that of the genes encoding ovine beta-lactoglobulin, human placental protein P14, rat alpha 2-urinary globulin, rat prostaglandin D synthase and human alpha 1-microglobulin, thus supporting the close evolutionary relationship of these genes. The 5'-noncoding region of LCN1 contains, besides a TATA and CAAT box, several motifs that resemble regulatory elements of other eukaryotic genes, including potential metal-responsive elements (MRE) and a cAMP-responsive element (CRE). As a basis for further investigations concerning the structure-function relationship and to generate a source of recombinant protein for X-ray crystallography studies, LCN1 was produced in Escherichia coli as a fusion with maltose-binding protein.

Assignment of the gene for human tear prealbumin (LCN1), a member of the lipocalin superfamily, to chromosome 8q24.

Tear prealbumin, a major protein of the human tear fluid, is also present in several other human body secretions. Recently it was shown to be a member of the lipocalin superfamily, a class of proteins that function as carriers for small hydrophobic molecules. Using a genomic tear prealbumin probe, we have mapped the gene (LCN1) to human chromosome 8q24 by fluorescent in situ hybridization.

cDNA cloning and sequencing reveals human tear prealbumin to be a member of the lipophilic-ligand carrier protein superfamily.

The gene encoding human tear prealbumin, a major component of the protein fraction of tear fluid, was cloned from total cDNA of lacrimal gland by polymerase chain reaction using synthetic oligonucleotides derived from N-terminal amino acid sequences of the purified protein. Sequence analysis and a computer-assisted homology search revealed this protein to be a member of the lipocalin superfamily, consisting of hydrophobic-ligand carriers. The deduced amino acid sequence of tear prealbumin shares 58% identity with von Ebner's gland protein from rat, which is supposed to be involved in taste reception. In addition, significant homology has also been found with other members of the lipocalins, e.g. with beta-lactoglobulin. The predicted secondary structure of tear prealbumin resembles that of beta-lactoglobulin in the number and positions of nine beta-sheets and one alpha-helix at the C-terminal part of the protein, thus indicating a three-dimensional structure similar to beta-lactoglobulin. Protein sequencing revealed that the observed electrophoretic heterogeneity of tear prealbumin is due to subtle differences at the N terminus of the protein. The function of tear prealbumin as a lipophilic carrier was further supported by the fact that it binds [3H]retinol in vitro. Although this protein was originally described to be tear-specific, a tear prealbumin-specific antiserum also reacted with proteins of human saliva, sweat, and nasal mucus.