Mutational analysis of the PRYSPRY domain of pyrin and implications for familial mediterranean fever (FMF)

Familial Mediterranean fever (FMF) is an autosomal, recessively inherited disease, characterized by recurrent fever and serositis that affects mainly patients of the Mediterranean basin. The gene responsible for FMF, named MEFV, was cloned and several missense mutations were found to be responsible for the disease. Based on a recent molecular analysis of MEFV gene mutations in 43 patients from Crete aiming to correlate specific genotypes and clinical manifestations of FMF, we were prompted to construct a three-dimensional model (3-D model) of the PRYSPRY domain of pyrin. The majority of the known MEFV mutations located on this domain have been classified, according to disease severity, and localized on this 3-D model. The functional consequences of these mutations and their implications on disease severity are discussed. Moreover, we report a putative novel missense mutation, S702C, which we identified in exon 10 of the MEFV gene and localized on the constructed 3-D model.     

Complex formation dynamics of native and mutated pyrin's B30.2 domain with caspase‐1

Pyrin protein is the product of the MEFV gene, mutations in which cause manifestation of familial Mediterranean fever (FMF). Functions of pyrin are not completely clear. The secondary structure of the pyrin is represented with four domains and two motifs. Mutations p.M680I, p.M694V, p.M694I, p.K695R, p.V726A, and p.A744S, which are located in the B30.2 domain of pyrin protein, are responsible for manifestation of the most common and severe forms of FMF. All the domains and the motifs of pyrin, are directly or indirectly, involved in the protein–protein interaction with proteins of apoptosis and regulate the cascade of inflammatory reactions, which is impaired due to pyrin mutations. It is well known, that malfunction of the pyrin‐caspase‐1 complex is the main reason of inflammation during FMF. Complete tertiary structure of pyrin and the effects of mutations in it are experimentally not studied yet. The aim of this study was to identify possible effects of the abovementioned mutations in the B30.2 domain tertiary structure and to determine their potential consequences in formation of the B30.2‐caspase‐1 complex. Using in silico methods, it was found, that these mutations led to structural rearrangements in B30.2 domain tertiary structure, causing shifts of binding sites and altering the interaction energy between B30.2 and caspase‐1.     

Screening of common and novel familial mediterranean fever mutations in south-east part of Turkey

Familial mediterranean fever (FMF) is an autosomal recessive autoinflammatory disorder (MIM# 249100), particularly common in populations of Mediterranean extraction. MEFV gene, responsible for FMF, encoding pyrin has recently been mapped to chromosome 16p13.3. In the present study, 3,341 unrelated patients with the suspicion of FMF in south-east part of Turkey between the years 2009 and 2013 were enrolled and genomic sequences of exon 2 and exon 10 of the MEFV gene were scanned for mutations by direct sequencing. We identified 43 different type of mutations and 9 of them were novel. DNA was amplified by PCR and subjected to direct sequencing for the detection of MEFV gene mutations. Among the 3,341 patients, 1,598 (47.8 %) were males and 1,743 (52.1 %) were females. The mutations were heterozygous in 806 (62.3 %), compound heterozygous in 188 (14.5 %), homozygous in 281 (21.8 %) and mutations had complex genotype in 17 (1.32 %) patients. No mutation was detected in 2,051 (61.4 %) patients. The most frequent mutations were M694V, E148Q, M680I(G/C) and V726A. We could not find any significant differences between the two common mutations according to the gender. Molecular diagnosis of MEFV is a useful tool in clinical practice, thus a future study relating to genotype/phenotype correlation of FMF in more and larger group in Turkish population involving the whole MEFV gene mutations is necessary.     

Gene encoding the mouse sulphamidase: cDNA cloning, structure, and chromosomal mapping

Sulphamidase is an exoglycosidase involved in the degradation of heparan sulfate. Lack of sulphamidase activity leads to the lysosomal storage disorder Mucopolysaccharidosis type IIIA (Sanfilippo type A OMIM No. 252900). At present there are no naturally occurring small animal models of this disease that could be of fundamental importance to study the pathophysiology of the disease and to try therapeutic strategies. Cloning of the mouse gene is an important step to create a mouse model for this common mucopolysaccharidosis. We have isolated and sequenced the gene encoding mouse sulphamidase. Comparison of the deduced amino acid sequences of human and mouse sulphamidase showed 88% identity and 93% similarity. The exon-intron structure of the gene has been determined with the mouse 10-kb gene divided in 8 exons. The mouse sulphamidase gene (Sgsh) was mapped to the distal end of Chromosome (Chr) 11, in a region that is homologous with a segment of human Chr 17 containing the orthologous human gene. Received: 26 July 1999 / Accepted: 3 February 2000     

Origin of structural domains of the serum-albumin gene family and a predicted structure of the gene for vitamin D-binding protein

We have recently determined complete DNA sequences for the human albumin and alpha-fetoprotein [AFP] genes and thus have identified their detailed structures. Each is composed of three domains of four exons, three of which are internal and one of which is a domain-linking exon. Equivalent exons in each domain show sufficient sequence and structural similarity to be considered homologous; additional unique exons at each end of the gene show no similarity to the internal triplicated structures. Since earlier, conflicting evolutionary models were based on analysis of single gene structures, we derived from five genes a series of consensus sequences representing the three internal exons as well as the domain-linking exon. The five genes were human and rat albumin and human, mouse, and rat AFP genes. Structurally equivalent exons of the different domains are shown to have arisen from a single exon in a one-domain precursor. Exons that bridge the domains arose from an unequal crossover that fused two exons of the precursor. Our model suggests that part of the coding sequence of the one-domain precursor may have been derived from an intron, by way of loss of a splice site. The consensus sequences were used to propose an intron-exon structure for the related gene encoding the serum vitamin D-binding protein (DBP). DBP is truncated relative to albumin and AFP, and we submit that this results from deletion of two internal exons in the third domain of the gene rather than from premature termination of the coding sequence.     

Isolation and characterization of the mouse ubiquitin-specific protease Usp15

We have characterized the mouse ortholog of the human ubiquitin-specific protease USP15. Mouse Usp15 consists of 981 amino acids with a predicted molecular mass of 112 kDa, contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes, and is 98% identical/99% similar to human USP15. Usp15 shares 59.5% identity/75.5% sequence similarity with the mouse Unp(Usp4) oncoprotein. Recombinant Usp15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to glutathione S-transferase. Usp15 can also cleave the ubiquitin-proline bond, as can USP15 and Usp4. Alignment of mouse and human Usp15 and Usp4 protein sequences suggested that Usp15/USP15 may be alternately spliced in a manner analogous to Usp4. Sequence analysis of RT-PCR products from several human and mouse cell lines and tissues revealed alternate splicing in all cells studied. Northern blot analysis of both mouse and human Usp15 revealed two differently sized mRNAs in all tissues examined, owing to alternate polyadenylation sites spaced by 1.5 kb. Chromosomal mapping by interspecific backcross analysis localized the Usp15 gene to the distal region of mouse Chromosome (Chr) 10. This region is syntenic with human Chr 12q24, the location of human USP15, and a different location to Unp(Usp4) (Chr 9). Identification of the mouse Usp15 gene (>69.5 kb) and human USP15 gene (145 kb) sequences in genome databases reveals that both are composed of 22 exons with identical splice sites, and both have an exon/intron structure identical to the mouse Usp4 gene, including the alternately spliced exon. Phylogenetic studies suggest that a sequence currently identified as a chicken Usp4 ortholog is in fact a USP15 ortholog, while bona-fide chicken, cow, and rat Usp4 orthologs can be identified in EST databases.     

Molecular cloning,expression pattern and chromosomal mapping of pig CD69

CD69 is a type II membrane protein belonging to C-type lectin family receptor, and expressed on activated leukocytes. Pig CD69 was cloned by RT-PCR using degenerate primers. Pig CD69 cDNA contains a 600 bp open reading frame with its predicted polypeptide sequence of 200 amino acids. Pig CD69 has 75%, 67%, and 57% sequence identity with cow, human, and mouse CD69, respectively. A splicing isoform, which lacks exon 2 encoding the transmembrane domain, was detected. Pig CD69 gene is located on Chromosome (Chr) 5q25 where the NKG2D gene was mapped. In RT-PCR analysis, pig CD69 mRNA was detected in activated PBL, NK cells, macrophages, monocytes, and granulocytes, but not in resting cells. The inducers for CD69 gene expression were PMA, PHA, LPS, G7 mAb, PNK-E mAb, PM16-6 mAb and the K562 cell line. Moreover, CD69 mRNA is expressed in bone marrow, spleen, thymus and lymph nodes but not in muscle, mammary gland, or the pig kidney cell line (LLC-PK(1)). These results indicate that pig Chr 5q25 contains the NK gene complex and CD69 can be used as an activation marker in pig cells of innate as well as acquired immune systems.     

Genomic organization of the human gamma adducin gene

We report the genomic structure of the human gamma adducin gene (ADD3). Adducin is a protein involved in cytoskeletal assembly and composed of alpha-beta or alpha-gamma subunits which share a high degree of homology between human and rat. Mutations in alpha subunit have been shown associated to both human and rat hypertension. The human ADD3 gene spans over 20 kb and is composed of at least 13 introns and 14 exons covering the entire coding region. The exon size ranges from 81 bp to greater than 293 bp and the intron size from 111 bp to longer than 3.2 kb. We also demonstrate the presence of an alternative splicing event around exon 13, whose sequence, position, and expression is analogous in rat Add3 gene. Moreover, human ADD3 amino acid sequence presents 91.9% of identity compared to rat sequence. Characterization of human ADD3 gene provides an important tool for mutation analysis.     

Interaction between pyrin and the apoptotic speck protein (ASC) modulates ASC-induced apoptosis

Patients with familial Mediterranean fever suffer sporadic inflammatory attacks characterized by fever and intense pain (in joints, abdomen, or chest). Pyrin, the product of the MEFV locus, is a cytosolic protein whose function is unknown. Using pyrin as a "bait" to probe a yeast two-hybrid library made from neutrophil cDNA, we isolated apoptotic speck protein containing a caspase recruitment domain (CARD) (ASC), a proapoptotic protein that induces the formation of large cytosolic "specks" in transfected cells. We found that when HeLa cells are transfected with ASC, specks are formed. After co-transfection of cells with ASC plus wild type pyrin, an increase in speck-positive cells is found, and speck-positive cells show increased survival. Immunofluorescence studies show that pyrin co-localizes with ASC in specks. Speck localization requires exon 1 of pyrin, but exon 1 alone of pyrin does not result in an increase in the number of specks. Exon 1 of pyrin and exon 1 of ASC show 42% sequence similarity and resemble death domain-related structures in modeling studies. These findings link pyrin to apoptosis pathways and suggest that the modulation of cell survival may be a component of the pathophysiology of familial Mediterranean fever.     

Mouse uroporphyrinogen decarboxylase: CDNA cloning,expression, and mapping

Uroporphyrinogen decarboxylase (URO-decarboxylase; EC 4.1.1.37), the heme biosynthetic enzyme responsible for the conversion of uroporphyrinogen III to coproporphyrinogen III, is the enzymatic defect in porphyria cutanea tarda, the most common porphyria. The mouse URO-decarboxylase cDNA was isolated from a mouse adult liver cDNA library. The longest clone of 1.5 kb, designated pmUROD-1, had 5′ and 3′ untranslated sequences of 281 and 97 bp, respectively, and an open reading frame of 1104 bp encoding a 367-amino acid polypeptide with a predicted molecular mass of 40,595 Da. The mouse and human coding sequences had 87.8% and 90.0% nucleotide and amino acid identity, respectively. The authenticity of the mouse cDNA was established by expression of the active enzyme in Escherichia coli. In addition, the analysis of two sets of multilocus genetic crosses localized the mouse gene, Urod, on Chromosome (Chr) 4, consistent with the map location of the human gene to a position of conserved synteny on Chr 1. The availability of the mouse URO-decarboxylase should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of this inherited porphyria. Received: 27 November 1995 / Accepted: 17 January 1996     

Identification and characterization of the mouse MutS homolog 5: Msh5

We have identified and characterized the complete cDNA and gene for the mouse MutS homolog 5 (Msh5), as a step toward understanding the molecular genetic mechanisms involved in the biological function of this new MutS homologous protein in mammals. The Msh5 cDNA contains a 2502-bp open reading frame (ORF) that encodes an 833-amino acid protein with a predicted molecular weight of 92.6 kDa, which shares 89.8% amino acid sequence identity with the human hMSH5 protein. Northern blot analysis demonstrated the presence of a Msh5 mRNA approximately 2.9-kb in length, most abundantly expressed in mouse testis. Yeast two-hybrid analysis indicated that the mouse Msh5 protein positively interacted with the human hMSH4 protein—suggesting that Msh5 shares common functional properties with its human counterpart. Sequence and structural analyses show that the mouse gene Msh5 spans approximately 18 kb and contains 24 exons that range in length from 36 bp for exon 7 to 392 bp for exon 1. Structural comparison with the human hMSH5 gene revealed that all of the Msh5 internal exons, but not introns, are conserved in length with the human hMSH5. The Msh5 gene is located on mouse Chromosome (Chr) 17 in a location that is syntenic to the region of human Chr 6 harboring the hMSH5 gene. The identification and characterization of Msh5 will facilitate studies of the potential functional roles of this new member of the MutS family. Received: 11 May 1999 / Accepted: 16 July 1999