ABCMdb

PMID: 20052366

Gee HY, Kim CK, Kim SW, Lee JH, Kim JH, Kim KH, Lee MG
The L441P mutation of cystic fibrosis transmembrane conductance regulator and its molecular pathogenic mechanisms in a Korean patient with cystic fibrosis.
J Korean Med Sci. 2010 Jan;25(1):166-71. Epub 2009 Dec 26., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Leu441Pro The genetic analysis of patient`s CFTR gene and the related molecular functional study revealed that the patient had a pathogenic L441P mutation in one allele. Login to comment 14 ABCC7 p.Leu441Pro J Korean Med Sci 2010; 25: 166-71 ISSN 1011-8934 DOI: 10.3346/jkms.2010.25.1.166 The L441P Mutation of Cystic Fibrosis Transmembrane conductance Regulator and its Molecular Pathogenic Mechanisms in a Korean Patient with Cystic Fibrosis Cystic fibrosis (CF) is an autosomal recessive disorder usually found in populations of white Caucasian descent. Login to comment 21 ABCC7 p.Leu441Pro After a comprehensive search for mutations in the CFTR gene, the patient was found to carry the non-synonymous L441P mutation in one allele. Login to comment 22 ABCC7 p.Leu441Pro ABCC7 p.Leu441Pro Molecular physiologic analysis of the L441P mutation of CFTR revealed that the L441P mutation completely abolished the CFTR Cl-channel activity by disrupting proper protein folding and membrane trafficking of CFTR protein. Login to comment 23 ABCC7 p.Leu441Pro These results confirmed the pathogenicity of the L441P mutation of CFTR circulating in the Korean population. Login to comment 46 ABCC7 p.Leu441Pro Interestingly, a non-synonymous L441P mutation of CFTR was identified in one allele by DGGE and consecutive nucleotide sequencings (Fig. 2). Login to comment 49 ABCC7 p.Leu441Pro However, no CFTR mutations including L441P were found in the blood samples from patient`s mother (Fig. 2). Login to comment 50 ABCC7 p.Leu441Pro Therefore, the L441P mutation was assumed to come from patient`s father. Login to comment 51 ABCC7 p.Leu441Pro It has been reported that the L441P mutation of CFTR is associated with CF in Japan and its allele frequency was estimated around 4.5% among CF patients in Japan (5). Login to comment 52 ABCC7 p.Leu441Pro However, the molecular pathogenic mechanisms of the L441P mutation of CFTR are currently unknown. Login to comment 53 ABCC7 p.Leu441Pro Therefore, we investigated the disease-causing mechanism of L441P using Fig. 1. Login to comment 58 ABCC7 p.Leu441Pro ABCC7 p.Leu441Pro A B A B Patient A WT Band C Lysate Blot: anti-CFTR Ab (M3A7) L441P WT Mother Exon 9 (Ex9.2) L441P 250 CTG → CCG: L441PPatient Forward Reverse T/C A/G T A Forward Reverse Mother Fig. 2. Login to comment 62 ABCC7 p.Leu441Pro (B) Nucleotide sequencing shows that the patient`s CFTR gene contains a mutation changed from T nucleotide at 1454 to C (heterozygous for L441P, CTG:Leu → CCG; Pro). Login to comment 63 ABCC7 p.Leu441Pro ABCC7 p.Leu441Pro KDa 160 105 Biotinylation Band B B WT Lysate Blot: anti-calnexin Ab L441P WT L441P 105 KDa 75 Biotinylation Fig. 3. Login to comment 64 ABCC7 p.Leu441Pro Immunoblotting and surface biotinylation of L441P mutant CFTR protein. Login to comment 65 ABCC7 p.Leu441Pro HEK 293 cells were transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation and protein samples were blotted with anti-CFTR M3A7 antibody (Cell Signaling Technology, Danvers, MA). Login to comment 66 ABCC7 p.Leu441Pro (A) Most of the wild type CFTR protein was detected as the fully glycosylated mature form (band C), whereas virtually all of the L441P mutant protein appeared as the core-glycosylated form of around 150 kDa (band B). Login to comment 71 ABCC7 p.Leu441Pro The CFTR plasmid carrying a L441P mutation was constructed by site-directed mutagenesis using QuickChange kit (Stratagene, La Jolla, CA, USA) with pCMV-CFTR plasmid according to the manufacturer`s protocol. Login to comment 73 ABCC7 p.Leu441Pro The total amount of CFTR protein was compared in HEK 293 cells transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation by immunoblotting of total cell lysates. Login to comment 76 ABCC7 p.Leu441Pro In immunoblotting of cell lysates, most of the wild type CFTR protein was detected as the fully glycosylated mature form, whereas virtually all of L441P mutant proteins appeared as the ER core-glycosylated form of about 150 kDa, also known as band B (Fig. 3). Login to comment 77 ABCC7 p.Leu441Pro These results imply that the L441P mutant CFTR protein has a defect in the ER-to-Golgi trafficking of the secretory pathway of membrane protein. Login to comment 78 ABCC7 p.Leu441Pro Consequently, the surface biotinylation results revealed that the L441P mutant protein failed to reach the plasma membrane, whereas the fully glycosylated form of wild type CFTR was expressed on the cell surface (Fig. 3). Login to comment 79 ABCC7 p.Leu441Pro To investigate the intracellular localizations of the mutant CFTR, immunostaining was performed in HEK 293 cells transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation. Login to comment 83 ABCC7 p.Leu441Pro On the other hand, L441P mutant protein was found in the ER, which was confirmed by co-localization with calnexin, an ER membrane protein (Fig. 4). Login to comment 89 ABCC7 p.Leu441Pro However, the cAMP treatment (5 μM FSK) failed to activate the Cl- currents in cells transfected with L441P mutant CFTR (Fig. 5). Login to comment 95 ABCC7 p.Leu441Pro ABCC7 p.Leu441Pro Immunocytochemistry of L441P mutant CFTR. HEK 293 cells were transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation, immunostained with anti-CFTR 24-1 antibody (R&D Systems) and fluorescein isothiocyante-conjugated secondary antibodies. Login to comment 98 ABCC7 p.Leu441Pro Wild type Calnexin Merge L441P Calnexin Merge ease progression. Login to comment 115 ABCC7 p.Gln1352His Some of them, especially E117G and Q1352H, showed an association with the monosymptomatic bronchiectasis or chronic pancreatitis. Login to comment 118 ABCC7 p.Leu441Pro A thorough examination on the coding regions and exon-intron splicing junctions revealed the L441P non-synonymous mutation in one allele. Login to comment 128 ABCC7 p.Leu441Pro ABCC7 p.Leu441Pro ABCC7 p.Leu441Pro ABCC7 p.Leu441Pro Fig. 5. cAMP-activated Cl-channel activity of L441P mutant CFTR. HEK 293 cells were transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation and the cAMP-activated Cl-channel activity was measured in the whole cell configuration. Login to comment 130 ABCC7 p.Leu441Pro Mean currents were normalized as current densities (pA/pF, n=6 for wild type CFTR and n=10 for L441P mutant CFTR). Login to comment 132 ABCC7 p.Leu441Pro ABCC7 p.Leu441Pro Currentdensity(pA/pF) 45 40 35 30 25 20 15 10 5 0 2,500 2,000 1,500 1,000 500 -500 -1,000 -1,500 -2,000 -2,500 WT L441P 20 40 60 80 100 120 140 P <0.01 Patch clamp I-V curve pA mV A B -140 -120-100 -80 -60 -40 -20 L441P WT Defects in the protein folding and processing during the secretory pathway of membrane protein are the major molecular pathogenic mechanisms of mutant CFTR (4). Login to comment 134 ABCC7 p.Leu441Pro The present study is the first study that substantially demonstrated the pathogenicity of the L441P mutation. Login to comment 135 ABCC7 p.Leu441Pro An integrated molecular physiologic examination revealed that the L441P mutation causes a processing defect. Login to comment 136 ABCC7 p.Leu441Pro ABCC7 p.Leu441Pro The L441P mutant protein was not fully glycosylated, which implies that the L441P mutant protein can not travel to the Golgi-complex and subsequently to the membrane. Login to comment 137 ABCC7 p.Leu441Pro This was verified by the absence of L441P mutant protein in the plasma membrane in immunostatining and surface biotinylation experiments. Login to comment 138 ABCC7 p.Leu441Pro Consequently, the cAMP-activated chloride channel activities of CFTR were completely deteriorated by the L441P mutation. Login to comment 140 ABCC7 p.Gly551Asp However, analysis of the CFTR haplotype structure revealed that significant proportions of Koreans have minor CFTR mutants, although the major disease causing alleles, such as DF508 and G551D, are extremely rare (10). Login to comment 141 ABCC7 p.Glu217Gly ABCC7 p.Gln1352His The heterozygote frequency of mild mutations, such as E217G and Q1352H, was estimated to 0.51% in the Korean population. Login to comment 164 ABCC7 p.Gln220* ABCC7 p.Gln98Arg Report of a Korean patient with cystic fibrosis, carrying Q98R and Q220X mutations in the CFTR gene. Login to comment