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To explore the biological and clinical implications of the structure/function relationships in factor XIII, mutations in two patients with type II deficiency were identified and characterized in a mammalian expression system. Nucleotide sequence analysis of the A subunit gene showed that case no. 1 had a deletion of 4 bp (AATT) in exon XI and that, in case no. 2, Gly562 (GGG) had been replaced by Arg(AGG). The deletion in case no. 1 leads to a premature termination at codon 464. Restriction digestion of amplified DNAs confirmed that both cases were homozygous for their respective mutations. Reverse transcription-polymerase chain reaction analysis demonstrated that the level of mRNA was greatly reduced in case no. 1, whereas the level of mutant mRNA expressed in case no. 2 was normal. Molecular modeling calculated that Arg562 changed the conformation of the A subunit, suggesting misfolding and/or destabilization of the molecule. To determine how these mutations impaired synthesis of the A subunit, recombinant A subunits bearing the mutations were expressed in mammalian cells. Pulse-chase experiments showed that the mutants were synthesized normally but disappeared rapidly, whereas the wild-type remained. These results indicate that both mutant proteins with an altered conformation become prone to rapid degradation, resulting in factor XIII deficiency in these patients.

Summary.To explore the implications of the structure/ function relationships in factor XIII, a patient with severe A subunit deficiency was examined at the DNA and RNA levels. Nucleotide sequence analysis of the patient's DNA amplified by PCR revealed that the patient had a replacement of C by T in the codon for Arg260. RT-PCR analysis demonstrated that only one kind of mRNA coding for the Arg260-Cys mutation was expressed in the patient at a normal level. Another possible defective allele of the A subunit gene with a G-A polymorphism was not expressed (null allele). The substitution of Arg260 by Cys located on the interface of two A subunits would preclude the reciprocal ionic interaction (salt bridge) between Arg260 and Asp404. Molecular modelling and, for the first time, molecular mechanics calculated that Cys260 changed the local conformation of the A subunit and reduced the electrostatic interaction between two monomers, suggesting destabilization of the molecule's dimer.Keywords: bleeding disorder, factor XIII deficiency, point mutation, molecular modelling, molecular mechanics.Coagulation factor XIII is a plasma transglutaminase consisting of two catalytic A and two non-catalytic B subunits (Folk & Chung, 1975;Lorand et al, 1980;Ichinose, 1995). A combination of cDNA cloning and amino acid sequence analysis established the primary structures of both subunits (Ichinose et al, 1986a, b). The A subunit contains an active site Cys314, and the B subunit is composed of 10 tandem repeats termed 'Sushi domains' . The gene for the A subunit of factor XIII is located on chromosome 6, and that for the B subunit is located on chromosome 1 (Board et al, 1988;Webb et al, 1989;Ichinose & Kaetsu, 1993). We previously characterized the genes for both the A and B subunits of human factor XIII (Ichinose & Davie, 1988;Bottenus et al, 1990). Determination of the genomic organization and sequence for both subunits has made it possible to characterize factor XIII deficiency at the DNA level (Ichinose & Kaetsu, 1993).Factor XIII deficiency has been classified into two categories (Girolami et al, 1978): type 1 deficiency, characterized by the lack of both the A and B subunits: and type II deficiency, characterized by the lack of the A subunit alone. To clarify the genetic bases of these diseases, two original cases of the type 1 deficiency were examined at the DNA level , as well as a patient with complete B subunit deficiency in whom the A subunit was also severely reduced (Hashiguchi et al, 1993). As a result, it has been concluded that both type 1 deficiency and B subunit deficiency are caused by genetic defects in the gene for the B subunit (Hashiguchi & Ichinose, 1995;Ichinose et al, 1996). Mutations in the gene for the A subunit have also been identified in patients with A subunit deficiency (or former type II factor XIII deficiency) (Kamura et al, 1992;Board et al, 1992;Ichinose & Kaetsu, 1993; Standen & Bowen, 1993;Mikkola et al, 1994Mikkola et al, , 1996Aslam et al, 1995Aslam et al, , 1997Anwar et al, 1995;Vreken et al, ...

To determine ligand-binding sites of a platelet-activating factor (PAF) receptor, alanine-scanning mutagenesis was carried out. All 23 polar amino acids in the putative 7-transmembrane (TM) domains of a guinea pig PAF receptor were individually replaced with alanine. The ligand-binding properties of mutant receptors were determined after transient expression in COS-7 cells. Mutants in TM II (N58A, D63A), TM III (N100A, T101A, S104A) and TM VII (D289A) displayed higher PAF-binding affinities than seen with the wild-type receptor. In contrast, mutants in TM V (H188A), TM VI (H248A, H249A, Q252A), and TM VII (Q276A, T278A) showed lower affinities. Representative mutants were then stably expressed in Chinese hamster ovary cells to observe PAF-induced cellular signals (arachidonate release, phosphatidylinositol hydrolysis, adenylyl cyclase inhibition). An N100A mutant with the highest affinity was constitutively active and was responsive to lyso-PAF, an inactive derivative of PAF. One nanomolar PAF induced no signals in low affinity mutants, an EC 50 value for the wild-type receptor. Three histidines (His-188, His-248, His-249) might form a binding pocket for the phosphate group of PAF, since zinc effectively inhibited ligand binding. Based on these results, a three-dimensional molecular model of PAF and its receptor was generated using bacteriorhodopsin as a reference protein.Platelet-activating factor (PAF), 1 is a potent phospholipid mediator with diverse physiological actions on a wide variety of cells and tissues. PAF is thought to play important roles in allergic disorders, inflammation, shock, and some diseases and also to have effects on the reproductive, cardiovascular, and central nervous systems (1-4). Despite the highly hydrophobic structure with a glycerophospholipid skeleton, PAF binds to a cell surface receptor, which was first cloned from a guinea pig lung cDNA library (5). The PAF receptor, with a seven-transmembrane (TM) topology like that of rhodopsin, belongs to a G protein-coupled receptor (GPCR) superfamily. PAF receptor homologs in four mammalian species (guinea pig, human, rat, and mouse) have been reported (6 -12). The PAF receptors couple with various second messenger systems including activation of phospholipase A 2 , C, and D; activation of mitogenactivated protein kinase, phosphatidylinositol 3-kinase, and tyrosine kinases; and inhibition of adenylyl cyclase, thus exerting pleiotropic effects (13-15).To date, a large body of information regarding ligand (agonist and antagonist)-binding characteristics of GPCRs represented by ␤-adrenergic receptors has been obtained (16), and this greatly contributes to effective clinical applications. Various PAF receptor antagonists have been developed as antiallergic and antiinflammatory drugs, but little is known of ligand-binding sites in the cloned PAF receptors. Most GPCR agonists are considered to bind to a hydrophobic core surrounded by seven-TM ␣-helices with electrostatic and hydrophobic force. We designed experiments using alanine-scanning muta...

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We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.

SummaryWe studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phel39Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Argl69Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.