Teruo Shimizu

Dystroglycan is a binding protein of laminin and merosin in peripheral nerve

α‐Dystroglycan, a 156 kDa dystrophin‐associated glycoprotein, binds laminin in skeletal muscle. Here we demonstrate that α‐dystroglycan is a binding protein of laminin (A/B1/B2) and merosin (M/B1/B2) in peripheral nerve. Immunocytochemical analysis demonstrates the localization of α‐dystroglycan and merosin surrounding myelin sheath of peripheral nerve fibers. Biochemical analysis demonstrates that the 120 kDa peripheral nerve α‐dystroglycan binds merosin as well as laminin. The binding of laminin and merosin is Ca2+ dependent and is inhibited by NACl and heparin. Recently, merosin was shown to be deficient in the peripheral nerve of dy mice which have defects in myelination. The interaction between α‐dystroglycan and merosin may play a role in the regulation of Schwann cell myelination and/or maintenance of myelin sheath.

Detection of O-mannosyl glycans in rabbit skeletal muscle α-dystroglycan

Abstract α-Dystroglycan, which is a cell surface component of dystroglycan complex, is known to bind laminin in basal lamina of muscle cells and Schwann cells. We found previously that a novel O-glycan, Siaα2-3Galβ1-4GlcNAcβ1-2Man, is the major oligosaccharide in bovine peripheral nerve α-dystroglycan, and that this structure might mediate the binding of laminin. In order to determine whether this structure is specific for peripheral nerve α-dystroglycan or present on different forms of α-dystroglycan, we analyzed the structures of the sialylated O-glycans of rabbit skeletal muscle α-dystroglycan. Their structures were elucidated to be a mixture of a core 1 O-glycan and the same O-mannosyl glycan that we found in bovine peripheral nerve. These results indicate that α-dystroglycan in different species and tissues share a common structure of its major O-linked acidic carbohydrate, suggesting its relevance to the basic functional role of α-dystroglycan.

Characterization of Dystroglycan‐Laminin Interaction in Peripheral Nerve

Abstract: Dystroglycan is encoded by a single gene and cleaved into two proteins, α‐ and β‐dystroglycan, by posttranslational processing. The 120‐kDa peripheral nerve isoform of α‐dystroglycan binds laminin‐2 comprised of the α2, β1, and γ1 chains. In congenital muscular dystrophy and dy mice deficient in laminin α2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan‐laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan‐laminin interaction in peripheral nerve. We demonstrate that (1) α‐dystroglycan is an extracellular peripheral membrane glycoprotein that links β‐dystroglycan in the Schwann cell outer membrane with laminin‐2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of α‐dystroglycan in the interaction with laminin.

Characterization of the Transmembrane Molecular Architecture of the Dystroglycan Complex in Schwann Cells

We have demonstrated previously 1) that the dystroglycan complex, but not the sarcoglycan complex, is expressed in peripheral nerve, and 2) that α-dystroglycan is an extracellular laminin-2-binding protein anchored to β-dystroglycan in the Schwann cell membrane. In the present study, we investigated the transmembrane molecular architecture of the dystroglycan complex in Schwann cells. The cytoplasmic domain of β-dystroglycan was co-localized with Dp116, the Schwann cell-specific isoform of dystrophin, in the abaxonal Schwann cell cytoplasm adjacent to the outer membrane. β-dystroglycan bound to Dp116 mainly via the 15 C-terminal amino acids of its cytoplasmic domain, but these amino acids were not solely responsible for the interaction of these two proteins. Interestingly, the β-dystroglycan-precipitating antibody precipitated only a small fraction of α-dystroglycan and did not precipitate laminin and Dp116 from the peripheral nerve extracts. Our results indicate 1) that Dp116 is a component of the submembranous cytoskeletal system that anchors the dystroglycan complex in Schwann cells, and 2) that the dystroglycan complex in Schwann cells is fragile compared with that in striated muscle cells. We propose that this fragility may be attributable to the absence of the sarcoglycan complex in Schwann cells.

Differential expression of dystrophin, utrophin and dystrophin-associated proteins in peripheral nerve.

The dystrophin‐glycoprotein complex is a novel laminin receptor in skeletal muscle. Dystrophin‐associated proteins are comprised of an extracellular glycoprotein of 156 kDa (156DAG), transmembrane glycoproteins of 50 kDa (50DAG), 43 kDa (43DAG) and 35 kDa (35DAG), and a cytoskeletal protein of 59 kDa (59DAP). The laminin‐binding 156DAG and 43DAG are encoded by a single gene and are now called α‐ and β‐dystroglycan, respectively. In neuromuscular junctions, utrophin, an autosomal homologue of dystrophin, is associated with sarcolemmal proteins identical or immunologically homologous to the dystrophin‐associated proteins. Here we demonstrate the co‐localization of Dp116 (a 116 kDa protein product of the DMD gene), full‐size utrophin, α‐ and β‐dystroglycan, 59DAP and 35DAG in a thin rim surrounding the outermost layer of myelin sheath of peripheral nerve fibers. The α‐dystroglycan in peripheral nerve had molecular weight of 120 kDa instead of 156 kDa, suggesting different levels of glycosylation between skeletal muscle and peripheral nerve. In sharp contrast to skeletal muscle, however, full‐size dystrophin and 50DAG were undetectable in peripheral nerve. Our results demonstrate the varied expression of the components of the dystrophin/utrophin‐glycoprotein complex between skeletal muscle and peripheral nerve suggesting the complex may exist in varied compositions and have varied functions in these two tissues.

Differential expression of the parkin gene in the human brain and peripheral leukocytes

Molecular cloning of the responsible gene on chromosome 6q25.2-27 for autosomal recessive juvenile parkinsonism (AR-JP) identified a novel protein of unknown function, named parkin. In patients with AR-JP, deletions most commonly involve exons 3-5 in the parkin gene. For mutation screening we tried to analyze the parkin transcript amplified by RT-PCR. Based on the assumption that illegitimate transcription of the parkin gene may occur in every cell type, we successfully amplified the parkin message from human peripheral leukocytes using RT-PCR. The parkin transcript in leukocytes was smaller in size than the full-length transcript in the brain. DNA sequencing determined that exons 3-5 were spliced out in the normal human leukocyte transcript. Our results demonstrate that alternative splicing produces distinct parkin transcripts in different tissues. Moreover, physiological splicing of deletion-prone exons may provide an important clue to understanding the pathogenesis of AR-JP.

Expression of dystroglycan and laminin-2 in peripheral nerve under axonal degeneration and regeneration

Abstract In Schwann cells, the transmembrane glycoprotein β-dystroglycan composes the dystroglycan complex, together with the extracellular glycoprotein α-dystroglycan which binds laminin-2, a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerve, the expression of β-dystroglycan and laminin-α2 chain was studied in rat sciatic nerves undergoing axonal degeneration and regeneration as well as in normal condition. In normal sciatic nerve, immunoreactivity for the cytoplasmic domain of β-dystroglycan was consistently and selectively localized in the Schwann cell cytoplasm underlying the outer (abaxonal) membrane apposing the basal lamina. While β-dystroglycan expression was gradually down-regulated in Schwann cells losing contact with axons during axonal degeneration, it was progressively up-regulated as the regenerating process of ensheathment and myelination proceeded during regeneration. Interestingly, β-dystroglycan expression, when detectable, was always restricted to the Schwann cell cytoplasm beneath the outer membrane apposing the basal lamina during both axonal degeneration and regeneration. Furthermore, laminin-α2 immunoreactivity roughly paralleled that of β-dystroglycan during both axonal degeneration and regeneration, indicating that the expression of β-dystroglycan and laminin-α2 is induced and maintained by the Schwann cell contact with axons. Our results indicate that the dystroglycan complex is involved in the adhesion of the Schwann cell outer membrane with the basal lamina and suggest that the dystroglycan complex may play a role in the process of Schwann cell ensheathment and myelination through the interaction with laminin-2.

Abnormal expression of laminin suggests disturbance of sarcolemma-extracellular matrix interaction in Japanese patients with autosomal recessive muscular dystrophy deficient in adhalin.

Dystrophin is associated with several novel sarcolemmal proteins, including a laminin-binding extracellular glycoprotein of 156 kD (alpha-dystroglycan) and a transmembrane glycoprotein of 50 kD (adhalin). Deficiency of adhalin characterizes a severe autosomal recessive muscular dystrophy prevalent in Arabs. Here we report for the first time two mongoloid (Japanese) patients with autosomal recessive muscular dystrophy deficient in adhalin. Interestingly, adhalin was not completely absent and was faintly detectable in a patchy distribution along the sarcolemma in our patients. Although the M and B2 subunits of laminin were preserved, the B1 subunit was greatly reduced in the basal lamina surrounding muscle fibers. Our results raise a possibility that the deficiency of adhalin may be associated with the disturbance of sarcolemma-extracellular matrix interaction leading to sarcolemmal instability.

Abnormal expression of heparan sulfate proteoglycan on basal lamina of muscle fibers in two Japanese patients with adhalin deficiency

We recently reported the selective reduction of the B1 subunit of laminin in two Japanese patients with adhalin deficiency. We here investigated immunohistochemically the expression of other components of the extracellular matrix (ECM), including collagen type IV, heparan sulfate proteoglycan can (HSPG), chondroitin-4-sulfate proteoglycan, decorin, and fibronectin in adhalin deficiency, compared with other types of muscular dystrophy. We found a reduction of HSPG on the basal lamina surrounding each muscle fiber in adhalin deficiency compared with HSPG in other diseases. This finding may be characteristic evidence of the disturbance of the sarcolemma-ECM interaction and the sarcolemmal instability in adhalin deficiency. Recently, a direct role of HSPG in fibroblast growth factor (FGF) signal transduction was demonstrated. Further investigation is required to determine if the dysfunction of FGF is relevant to the pathogenesis of adhalin deficiency.

Deficiency of a 180-kDa extracellular matrix protein in Fukuyama type congenital muscular dystrophy skeletal muscle

Abnormalities of the proteins constituting the extracellular matrix have been shown to play important roles in the molecular pathogenesis of muscular dystrophies. In the present study, we have established a monoclonal antibody against a human skeletal muscle extracellular matrix protein. The antibody M1 recognized a single 180-kDa protein (p180) by immunoblot analysis of normal human skeletal muscle and gave a strong and continuous signal along the sarcolemma by immunohistochemical analysis. Furthermore, p180 could be solubilized either under a strong alkaline condition, or in the presence of EDTA or detergents such as Triton X-100, indicating that p180 was an extracellular matrix protein. Interestingly, p180 was deficient in the skeletal muscle of the patients with Fukuyama-type congenital muscular dystrophy (FCMD), but not other muscular diseases, by both immunohistochemical and immunoblot analyses. We presume that the deficiency of p180 in FCMD is caused specifically by the primary deficiency of fukutin, the causative protein of FCMD, and plays an important role in muscle cell degeneration in this disease.