ABSTRACT
Genetic testing has made diagnosis and treatment possible for many infants. With the addition of many new tests over the past few years, it is important to understand the clinical usefulness of each of these tests. Selecting the correct method of genetic testing assists in obtaining an accurate diagnosis and development of a plan of care for the infant.
Subject(s)
Chromosome Aberrations , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/nursing , Genetic Testing/methods , Infant, Premature, Diseases/genetics , Infant, Premature, Diseases/nursing , Humans , In Situ Hybridization, Fluorescence , Infant, Newborn , Neonatal Screening , Oligonucleotide Array Sequence Analysis , Prenatal Diagnosis/nursingABSTRACT
CLCA proteins (calcium-activated chloride channel regulators) have been linked to diseases involving secretory disorders, including cystic fibrosis (CF) and asthma. They have been shown to modulate endogenous chloride conductance, possibly by acting as metalloproteases. Based on the differential processing of the subunits after posttranslational cleavage, two subgroups of CLCA proteins can be distinguished. In one subgroup, both subunits are secreted, in the other group, the carboxy-terminal subunit possesses a transmembrane segment, resulting in shedding of only the amino-terminal subunit. Recent data on the post-translational cleavage and proteolytic activity of CLCA are limited to secreted CLCA. In this study, we characterized the cleavage of mCLCA6, a murine CLCA possessing a transmembrane segment. As for secreted CLCA, the cleavage in the endoplasmic reticulum was not observed for a protein with the E157Q mutation in the HEXXH motif of mCLCA6, suggesting that this mutant protein and secreted CLCA family members share a similar autoproteolytic cleavage mechanism. In contrast to secreted CLCA proteins with the E157Q mutation, the uncleaved precursor of the mCLCA6E157Q mutant reached the plasma membrane, where it was cleaved and the amino-terminal subunit was shed into the supernatant. Using crude membrane fractions, we showed that cleavage of the mCLCA6E157Q protein is zinc-dependent and sensitive to metalloprotease inhibitors, suggesting secondary cleavage by a metalloprotease. Interestingly, anchorage of mCLCA6E157Q to the plasma membrane is not essential for its secondary cleavage, because the mCLCA6(Δ™)E157Q mutant still underwent cleavage. Our data suggest that the processing of CLCA proteins is more complex than previously recognized.
Subject(s)
Cell Membrane/metabolism , Chloride Channels/metabolism , Endoplasmic Reticulum/metabolism , Enterocytes/metabolism , Protein Precursors/metabolism , Proteolysis , Amino Acid Motifs , Amino Acid Substitution , Animals , Cell Membrane/enzymology , Chelating Agents/pharmacology , Chloride Channels/chemistry , Chloride Channels/genetics , Endoplasmic Reticulum/enzymology , Enterocytes/enzymology , HEK293 Cells , Humans , Metalloproteases/antagonists & inhibitors , Metalloproteases/metabolism , Mice , Mutagenesis, Site-Directed , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Structure, Tertiary , Zinc/pharmacologyABSTRACT
CLCA proteins represent a large family of proteins widely expressed in mammalian tissues with a unique expression pattern for each family member analyzed so far. However, their functions in normal and diseased tissues are poorly understood. Here, we present the cellular expression pattern of mCLCA5 in murine tissues using immunohistochemistry, confocal laser scanning microscopy and immune electron microscopy with specific antibodies and RT-qPCR following laser-capture microdissection. The mCLCA5 protein was localized to granular layer keratinocytes of virtually all stratified squamous epithelia of the body. Biochemical protein characterizations revealed that the amino-terminal cleavage product is fully secreted by the cell, while the carboxy-terminal cleavage product remains associated with the cell. The results imply that mCLCA5 may play a role in maturation and keratinization of squamous epithelial cells.
Subject(s)
Chloride Channels/genetics , Chloride Channels/metabolism , Epithelium/metabolism , Keratinocytes/metabolism , Animals , Chloride Channels/analysis , Epithelium/chemistry , Epithelium/ultrastructure , Female , Immunohistochemistry , Keratinocytes/chemistry , Keratinocytes/ultrastructure , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Microscopy, Immunoelectron , RNA, Messenger/analysis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Two new calcium-activated chloride channel (CLCA) family members, mCLCA5 and mCLCA6, have been cloned from mouse eye and intestine, respectively. mCLCA5 is highly homologous to hCLCA2, and mCLCA6 is highly homologous to hCLCA4. mCLCA5 is widely expressed with strong expression in eye and spleen, whereas mCLCA6 is primarily expressed in intestine and stomach. mCLCA6 is also expressed as a splice variant lacking exon 8 and part of exon 10 in intestine and stomach. Transfection of tsA201 cells with enhanced green fluorescent protein-tagged versions of the three cDNAs reveals protein products of 155 and 65 kDa for mCLCA5 and mCLCA6 and 145 and 65 kDa for the mCLCA6 splice variant. In vitro translation of mCLCA5 generates a 90-kDa protein that does not appear to be glycosylated. mCLCA6 also generates a 90-kDa protein that is glycosylated to a 110-kDa product, whereas the mCLCA6 splice variant generates an 80-kDa product that is 100 kDa after glycosylation. Treatment of enhanced green fluorescent protein-tagged mCLCA6 with PNGase F (peptide: N-glycosidase F) to remove N-linked glycosyl groups shows a reduction in size of the 65 kDa product to 60 kDa. Consistent with the hypothesis that mCLCA5, mCLCA6, and its splice variant encode calcium-activated chloride channels, in HEK293 cells expressing CLCAs ionomycin-evoked increases in intracellular calcium stimulated a current that reversed near Cl(-) equilibrium potential, E(Cl). Furthermore, these currents were inhibited by the chloride channel blocker niflumic acid. Given the prominent role of hCLCA2 in cancer cell adhesion and the unique high level of expression of hCLCA4 in brain, the identification of their murine counterparts presents the opportunity to clarify the role of CLCAs in disease and normal cell physiology.
Subject(s)
Chloride Channels/genetics , Chloride Channels/physiology , Eye/chemistry , Intestines/chemistry , Amino Acid Sequence , Animals , Cell Line , Chloride Channels/chemistry , Cloning, Molecular , Electrophysiology , Glycosylation , Mice , Molecular Sequence Data , Molecular Weight , Protein Isoforms , Sequence Alignment , TransfectionABSTRACT
In myotonic dystrophy (dystrophia myotonica, DM), expression of RNAs that contain expanded CUG or CCUG repeats is associated with degeneration and repetitive action potentials (myotonia) in skeletal muscle. Using skeletal muscle from a transgenic mouse model of DM, we show that expression of expanded CUG repeats reduces the transmembrane chloride conductance to levels well below those expected to cause myotonia. The expanded CUG repeats trigger aberrant splicing of pre-mRNA for ClC-1, the main chloride channel in muscle, resulting in loss of ClC-1 protein from the surface membrane. We also have identified a similar defect in ClC-1 splicing and expression in two types of human DM. We propose that a transdominant effect of mutant RNA on RNA processing leads to chloride channelopathy and membrane hyperexcitability in DM.
