Extra Cys Research Articles

Molecular cloning and sequence analysis of 1Dx-type HMW-GS genes from different wheat varieties

AbstractHigh-molecular-weight glutenin subunits (HMW-GS) contribute to dough elasticity and bread baking quality in wheat. In this study, wheat varieties were classified based on their HMW-GS composition into three groups: 1Dx5 (5 + 10, Gaoyou 8901, Xinmai 28, Xinmai 19, Xinmai 26 and Jinbaoyin), 1Dx2 (2 + 12, Zhoumai 24, Xinmai 9 and Yumai) and 1Dx4 (4 + 12, Aikang 58). Sequence analysis showed that 1Dx-GY8901, 1Dx-XM28, 1Dx-XM19 and 1Dx-XM26 were similar to the 1Dx5 gene and clustered on the same branch, while 1Dx-AK58, 1Dx-ZM24, 1Dx-JBY, 1Dx-YM, 1Dx-XM9 and 1Dx-JBY were more similar to the 1Dx2 gene and clustered on the same branch with 1Dx.2.2. There was a mutation of Ser to Cys at position S2, for an extra Cys in the repeat regions of 1Dx-XM19, 1Dx-XM26, 1Dx-XM28 and 1Dx-GY8901. The wheat HMW-GS genes exhibited similar percentages of α-helix, extended strand, β-turn and random coil structure, with ranges of 13.33–13.59, 4.77–5.78, 7.08–9.18 and 72.3–73.94%, respectively. Sequence conservation and the composition of HMW-GS subunits were also analysed for a series of strong gluten wheat varieties, Xinmai 9 (1, 7 + 8, 2 + 12), Xinmai 19 (1, 7 + 9, 5 + 10), Xinmai 26 (1, 7 + 8, 5 + 10) and Xinmai 28 (1, 7 + 9, 5 + 10). The results of this work should facilitate future breeding efforts and provide the theoretical basis for wheat quality improvement.

Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.

Characterization of an Atypical Metalloproteinase Inhibitors Like Protein (Sbp8-1) From Scallop Byssus.

Adhesion is a vital physiological process for many marine molluscs, including the mussel and scallop, and therefore it is important to characterize the proteins involved in these adhesives. Although several mussel byssal proteins were identified and characterized, the study for scallop byssal proteins remains scarce. Our previous study identified two foot-specific proteins (Sbp7, Sbp8-1), which were annotated as the tissue inhibitors of metalloproteinases (TIMPs). Evolutionary analysis suggests that the TIMP genes of Chlamys farreri had gone through multiple gene duplications during evolution, and their potential functional roles in foot may have an ancient evolutionary origin. Focusing on the Sbp8-1, the sequence alignment and biochemical analyses suggest that Sbp8-1 is an atypical TIMP. One significant feature is the presence of two extra free Cys residues at its C-terminus, which causes the Sbp8-1 polymerization. Considering the fact that the no inhibitory activity was observed and it is mainly distributed in byssal thread and plaque, we proposed that this atypical Sbp8-1 may play as the cross-linker in scallop byssus. This study facilitates not only the understanding of scallop byssus assembly, also provides the inspiration of water-resistant materials design.

Dietary supplementation with cysteine prevents adverse metabolic outcomes of repeated cures with paracetamol in old rats.

Cysteine (Cys), a conditionally indispensable amino acid, is required for the detoxification of paracetamol (acetaminophen, N-acetyl-para-aminophenol, 4-hydroxy-acetanilide, APAP), a drug of widespread use in older persons. We recently reported that repeated APAP cures could worsen sarcopenia in old rats, likely to be due to the impairment of Cys/GSH homoeostasis. The aim of the study was to evaluate whether a dietary Cys supplementation during APAP cures could improve Cys/GSH homoeostasis and thus preserve skeletal muscle. Male 21·5-month-old Wistar rats received three 2-week-long cures of APAP (1 % of diet) alone or with extra Cys (0·5 % of diet), intercalated with washout periods of 2 weeks (APAP and APAP-Cys groups, respectively). They were compared with untreated control rats (CT group). CT and APAP-Cys groups were pair-fed to the APAP group. Dietary Cys supplementation was efficient to prevent increase in liver mass (P<0·0001), decrease in liver GSH (P<0·0001), increase in blood GSH concentration (P<0·0001), and to some extent, decrease in plasma free Cys concentration (P<0·05), all induced by repeated APAP cures. The addition of Cys to APAP cures decreased plasma alanine transaminase (P<0·05), the fractional synthesis rate of liver proteins (P<0·01), and increased masses of extensor digitorum longus (P<0·01), and soleus (P<0·05), compared with the APAP group. Cys supplementation prevented alteration in Cys/GSH homoeostasis and increased some muscle masses in old rats under repeated cures with a non-toxic dose of APAP.

Evidence that glutathione and the glutathione system efficiently recycle 1-cys sulfiredoxin in vivo.

Typical 2-Cys peroxiredoxins (2-Cys Prxs) are Cys peroxidases that undergo inactivation by hyperoxidation of the catalytic Cys, a modification reversed by ATP-dependent reduction by sulfiredoxin (Srx). Such an attribute is thought to provide regulation of 2-Cys Prxs functions. The initial steps of the Srx catalytic mechanism lead to a Prx/Srx thiolsulfinate intermediate that must be reduced to regenerate Srx. In Saccharomyces cerevisiae Srx, the thiolsulfinate is resolved by an extra Cys (Cys48) that is absent in mammalian, plant, and cyanobacteria Srxs (1-Cys Srxs). We have addressed the mechanism of reduction of 1-Cys Srxs using S. cerevisiae Srx mutants lacking Cys48 as a model. We have tested the recycling of Srx by glutathione (GSH) by a combination of in vitro steady-state and single-turnover kinetic analyses, using enzymatic coupled assays, Prx fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reverse-phase chromatography coupled to mass spectrometry. We demonstrate that GSH reacts directly with the thiolsulfinate intermediate, by following saturation kinetics with an apparent dissociation constant of 34 μM, while producing S-glutathionylated Srx as a catalytic intermediate which is efficiently reduced by the glutaredoxin/glutathione reductase system. Total cellular depletion of GSH impacted the recycling of Srx, confirming in vivo that GSH is the physiologic reducer of 1-Cys Srx. Our study suggests that GSH binds to the thiolsulfinate complex, thus allowing non-rate limiting reduction. Such a structural recognition of GSH enables an efficient catalytic reduction, even at very low GSH cellular levels. This study provides both in vitro and in vivo evidence of the role of GSH as the primary reducer of 1-Cys Srxs.

Datura stramonium agglutinin: Cloning, molecular characterization and recombinant production in Arabidopsis thaliana

Datura stramonium seeds contain at least three chitin-binding isolectins [termed Datura stramonium agglutinin (DSA)] as homo- or heterodimers of A and B subunits. We isolated a cDNA encoding isolectin B (DSA-B) from an immature fruit cDNA library; this contained an open reading frame encoding 279 deduced amino acids, which was confirmed by partial sequencing of the native DSA-B peptide. The sequence consisted of: (i) a cysteine (Cys)-rich carbohydrate-binding domain composed of four conserved chitin-binding domains and (ii) an extensin-like domain of 37 residues containing four SerPro4-6 motifs that was inserted between the second and third chitin-binding domains (CBDs). Although each chitin-binding domain contained eight conserved Cys residues, only the second chitin-binding domain contained an extra Cys residue, which may participate in dimerization through inter-disulfide bridge formation. Using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, the molecular mass of homodimeric lectin composed of two B-subunits was determined as 68,821 Da. The molecular mass of the S-pyridilethylated B-subunit were found to be 37,748 Da and that of the de-glycosylated form was 26,491 Da, which correlated with the molecular weight estimated from the deduced sequence. Transgenic Arabidopsis plants overexpressing the dsa-b demonstrated hemagglutinating activity. Recombinant DSA-B was produced as a homodimeric glycoprotein with a similar molecular mass to that of the native form. Moreover, the N-terminus of the purified recombinant DSA-B protein was identical to that of the native DSA-B, confirming that the cloned cDNA encoded DSA-B.

Plant Thioredoxin CDSP32 Regenerates 1-Cys Methionine Sulfoxide Reductase B Activity through the Direct Reduction of Sulfenic Acid

Thioredoxins (Trxs) are ubiquitous enzymes catalyzing the reduction of disulfide bonds, thanks to a CXXC active site. Among their substrates, 2-Cys methionine sulfoxide reductases B (2-Cys MSRBs) reduce the R diastereoisomer of methionine sulfoxide (MetSO) and possess two redox-active Cys as follows: a catalytic Cys reducing MetSO and a resolving one, involved in disulfide bridge formation. The other MSRB type, 1-Cys MSRBs, possesses only the catalytic Cys, and their regeneration mechanisms by Trxs remain unclear. The plant plastidial Trx CDSP32 is able to provide 1-Cys MSRB with electrons. CDSP32 includes two Trx modules with one potential active site (219)CGPC(222) and three extra Cys. Here, we investigated the redox properties of recombinant Arabidopsis CDSP32 and delineated the biochemical mechanisms of MSRB regeneration by CDSP32. Free thiol titration and 4-acetamido-4'-maleimidyldistilbene-2,2'-disulfonic acid alkylation assays indicated that the Trx possesses only two redox-active Cys, very likely the Cys(219) and Cys(222). Protein electrophoresis analyses coupled to mass spectrometry revealed that CDSP32 forms a heterodimeric complex with MSRB1 via reduction of the sulfenic acid formed on MSRB1 catalytic Cys after MetSO reduction. MSR activity assays using variable CDSP32 amounts revealed that MSRB1 reduction proceeds with a 1:1 stoichiometry, and redox titrations indicated that CDSP32 and MSRB1 possess midpoints potentials of -337 and -328 mV at pH 7.9, respectively, indicating that regeneration of MSRB1 activity by the Trx through sulfenic acid reduction is thermodynamically feasible in physiological conditions.

Folding anomalies of neuroligin3 caused by a mutation in the α/β-hydrolase fold domain

Proteins of the α/β-hydrolase fold family share a common structural fold, but perform a diverse set of functions. We have been studying natural mutations occurring in association with congenital disorders in the α/β-hydrolase fold domain of neuroligin (NLGN), butyrylcholinesterase (BChE), acetylcholinesterase (AChE). Starting from the autism-related R451C mutation in the α/β-hydrolase fold domain of NLGN3, we had previously shown that the Arg to Cys substitution is responsible for endoplasmic reticulum (ER) retention of the mutant protein and that a similar trafficking defect is observed when the mutation is inserted at the homologous positions in AChE and BChE. Herein we show further characterization of the R451C mutation in NLGN3 when expressed in HEK-293, and by protease digestion sensitivity, we reveal that the phenotype results from protein misfolding. However, the presence of an extra Cys does not interfere with the formation of disulfide bonds as shown by reaction with PEG-maleimide and estimation of the molecular mass changes. These findings highlight the role of proper protein folding in protein processing and localization.

Molecular characterization of LMW-GS genes from a somatic hybrid introgression line II-12 between Triticum aestivum and Agropyron elongatum in relation to quick evolution

In order to exploit the evolution and find novel low-molecular-weight glutenin subunit (LMW-GS) for improvement of common wheat quality, thirteen variants from a somatic hybrid introgression line II-12 between Triticum aestivum cv. Jinan 177 (JN177) and Agropyron elongatum were characterized via genomic PCR. Four clones were pseudogenes because they contained an internal stop codon. The remaining nine variants contained intact open reading frames (ORFs). Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously. However, they have some unique modifications in the structures. For example, EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain. Both EU159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain, but not in the N-conserved domain traditionally. These structural alterations may have positive contributions to wheat flour quality. The results of phylogeny showed that most LMW-GS variances from II-12 were homologous to those from parent JN177 and other wheat lines. The reason for quick evolution of LMW-GS in II-12 was discussed.

MTPA: A crustacean metallothionein that affects hepatopancreatic mitochondrial functions

Metallothioneins are cysteine-rich proteins, with a high capacity to bind metallic ions, and for which a precise biological role has not been established. Here we investigated the effects of MTPA, a metallothionein from the lobster Panulirus argus, on mitochondrial oxygen consumption and ROS production. An HPLC-RP-ESI-MS analysis of recombinant MTPA showed that despite its extra Cys, MTPA binds 6 Zn2+ per molecule akin to other crustacean metallothioneins with 18 Cys. The extra Cys is not involved in zinc binding, since its side-chain would be oriented to the outside of the molecule according to a preliminary model of the tridimensional structure of MTPA. MTPA-Zn2+6 is imported into the hepatopancreatic mitochondria intermembrane space and inhibits mitochondrial oxygen consumption, increasing thereby ROS production. Nevertheless, the stimulation of ROS production by MT-bound Zn2+ is weaker compared to equivalent amounts of free Zn2+, suggesting that MTPA protects against oxidative stress. This constitutes the first report on metallothioneins effects on mitochondrial function in invertebrates and agrees with the results described for mammals, suggesting a connection between metallothioneins and energy metabolism.

Characterization of high‐molecular‐weight glutenin subunit genes fromElytrigia elongata

AbstractThree high‐molecular‐weight glutenin subunit (HMW‐GS) genes fromElytrigia elongata(Host) Nevski were characterized by determining the coding sequences of two x‐type subunit genesEe2.1andEe1.9, and one y‐type subunit geneEe1.8with 2082, 1938 and 1788 bp, respectively. The numbers of amino acids in the central repeat domains of Ee2.1, Ee1.9 and Ee1.8 were considerably fewer than the other known HMW‐GSs with substitutions, insertions and/or deletions involving a single or more amino acid residues. Moreover, an extra cysteine (Cys) was found in the repeated domain of x‐type subunit Ee2.1. The difference in the number and position of Cys residues might be associated with the good dough quality. The extra Cys in the good‐quality subunit Dx5 was at the beginning of the repetitive domain, while the extra Cys in Ee2.1 was at the central repetitive domain. Evolutionary relationships between the HMW‐GSs fromE. elongataand the known HMW‐GSs were estimated. It transpired that Ee1.9, Ee2.1 and Bx7 were clustered into one subgroup with high bootstrap values, while Ee1.8 was closely related to Ay.

LMW-GS genes in Agropyron elongatum and their potential value in wheat breeding

To study the usefulness of low-molecular-weight glutenin subunits (LMW-GS) of Agropyron elongatum (Host) Nevski to wheat (Triticum aestivum L.) quality improvement, we characterized LMW-GS genes of A. elongatum. Nine LMW-GS genes of A. elongatum, which were named AeL1 to AeL9, were cloned by genomic PCR. After sequencing, we obtained complete open reading frames from AeL2 to AeL8 and partial genes of AeL1 and AeL9. All nine sequences are homoeologous to those of wheat and related grasses. Comparison of the deduced amino acid sequences with those of published LMW-GS suggests that the basic structures of all the subunits are very similar. However, except for AeL4 and AeL5, which contain the identical N-terminal sequence with LMW-m, other LMW-GS sequences separated from A. elongatum cannot be classified according to previous criteria for the three types: LMW-m (methionine), LMW-s (serine), and LMW-i (isoleucine), and then 12 groups. In addition, there are some characters in the LMW-GS sequences of A. elongatum: AeL2, AeL3, and AeL6 involve a Cys residue in the signal peptide respectively, which is absent in most of LMW-GS; AeL3, AeL6, AeL8, and AeL9 start their first Cys residues in the N-terminal repetitive domains, respectively; both AeL2 and AeL5 have nine Cys residues, with an extra Cys residue in the N-terminal repetitive domain and the repetitive and glutamine-rich domain; AeL2, AeL3, AeL6, and AeL9 comprise long repetitive domains. Phylogenetic analysis indicates that there is a relatively weak sequence identity between the LMW-GS genes from A. elongatum cloned in this study and those reported from other plants. Three LMW-GS sequences, AeL2, AeL3, and AeL6, are clustered to Glu-A3 from wheat than to those from other plants. The possible use of these genes in relation to the high quality of hybrid wheat is discussed.

The two subfamilies of rice glutelin differ in both primary and higher-order structures

Rice glutelin, which accounts for 70–80% of the total proteins of the seeds, consists of two nutritionally different subfamilies (A and B types). Although the similarity in primary sequences between the two subfamilies is as high as 60%, we established conditions to discriminate the two subfamilies when low amounts of antigen are analyzed by immunoblot methods. The glutelin α polypeptides can be resolved into six bands labeled α1 to α6 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration analysis showed that glutelin exists as a polymerized and a smaller molecular weight form. Immunoblot analysis of SDS-PAGE resolved polypeptides showed that α2, α3, and α4 are an A type and that these A types as well as α1, a B type, are polymerized. The polymerization tendency clearly differed between the two subfamilies except for α1, which may be derived from GluB-4 as suggested by analysis using Escherichia coli expression systems of glutelin cDNA regions corresponding to α polypeptides. GluB-4 and all the A type subunits have an extra Cys residue in the hypervariable regions, corresponding to the C-terminal region of α polypeptide. Accordingly, the extra Cys residue is hypothesized to be responsible for the polymerization of glutelin.

The two subfamilies of rice glutelin differ in both primary and higher-order structures

Rice glutelin, which accounts for 70–80% of the total proteins of the seeds, consists of two nutritionally different subfamilies (A and B types). Although the similarity in primary sequences between the two subfamilies is as high as 60%, we established conditions to discriminate the two subfamilies when low amounts of antigen are analyzed by immunoblot methods. The glutelin α polypeptides can be resolved into six bands labeled α1 to α6 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration analysis showed that glutelin exists as a polymerized and a smaller molecular weight form. Immunoblot analysis of SDS-PAGE resolved polypeptides showed that α2, α3, and α4 are an A type and that these A types as well as α1, a B type, are polymerized. The polymerization tendency clearly differed between the two subfamilies except for α1, which may be derived from GluB-4 as suggested by analysis using Escherichia coli expression systems of glutelin cDNA regions corresponding to α polypeptides. GluB-4 and all the A type subunits have an extra Cys residue in the hypervariable regions, corresponding to the C-terminal region of α polypeptide. Accordingly, the extra Cys residue is hypothesized to be responsible for the polymerization of glutelin.

A positive charge preservation at position 116 of alpha A-crystallin is critical for its structural and functional integrity.

An autosomal dominant congenital cataract associated with a missense mutation, Arg-116 to Cys (R116C), in the coding sequence of human alphaA-crystallin has been reported. Subsequent study of this mutant, generated by site-directed mutagenesis, showed significant changes in secondary and tertiary structures, partial loss of chaperone activity, and substantially increased oligomeric size. The study presented here aims to show whether these changes are due to the loss of a positive charge at this position or due to the presence of an extra Cys. To show this, Arg-116 in alphaA-crystallin was mutated to Lys (R116K), Cys (R116C), Gly (R116G), and Asp (R116D) and expressed in Escherichia coli cells. The wild-type (alphaA-wt) and mutant proteins were purified by size exclusion chromatography and characterized by measurements of circular dichroism, intrinsic tryptophan fluorescence, and TNS fluorescence and by determination of molecular masses and chaperone function which was assessed as the ability to suppress target protein aggregation or enhance target protein refolding. Mutation of Arg-116 to a Cys or Gly showed very similar changes in structure, oligomerization, and chaperone function which suggest that the presence of this Cys per se is not the cause of the changes. The R116K mutant, on the other hand, had nearly the same structure, oligomeric size, and chaperone function as alphaA-wt, whereas the mutant with an acidic amino acid in this position, R116D, showed drastic changes in protein structure. Thus, a positive charge must be preserved at this position for the structural and functional integrity of alphaA-crystallin.

Milkfish ( Chanos chanos) growth hormone cDNA cloning and mRNA expression in embryos and early larval stages

In an attempt to understand growth regulation in milkfish, the milkfish growth hormone (GH) and its cDNA were characterized and the expression of GH mRNA in embryos and larvae was examined by RT-PCR. The milkfish GH was purified from an alkaline extract of the pituitary by reverse-phase high-performance liquid chromatography and detected as an immuno-positive protein with anti-salmon GH serum. The complete sequence of milkfish pre-GH was determined by cDNA cloning and nucleotide sequencing. On the basis of the N-terminal amino acid analysis of the native protein, the pre-GH was found to consist of a signal peptide of 22 amino acids and a mature protein of 188 amino acids. Milkfish GH shows higher amino acid sequence identity with GHs of carps (91–94%) and salmonids (70%) than with GHs of more advanced teleosts (<60%) in good accordance with its taxonomic position in teleosts. It has five half Cys residues, four of which are at positions homologous with those of other known GHs and the extra Cys with those of carp GHs. The molecular weight of milkfish GH was estimated to be 22 kDa, which is comparable to the theoretical value. This suggests that milkfish GH is a simple protein, although it has two potential N-glycosylation sites. Semiquantitative RT-PCR showed that GH mRNA expression was relatively weak in embryos and newly hatched larvae but was already strong in 2-day old and older larvae.

IgG2a and IgA Co-Expression by the Natural Autoantibody-producing Murine B Lymphoma T560

The T560 B lymphoma produces polyreactive IgG2a with the features of natural autoantibody. All T560 cells bear and secrete IgG2a but a small fraction spontaneously co-express IgA. Cells secreting IgA alone cannot be detected. IgA secretion is enhanced by interaction of T560 cells either with activated T cells and cognate antigen, or with LPS, but not with cytokines, including 1L-5 and TGF-fl IgA and IgG2a mRNAs have identical VI86.2, DFL 16 and JH1 sequences from framework 2 through JH1. PCR analysis reveals that previous recombination events have led to deletion of the μ., γ3, γl, γ2b constant region genes from both the productive and the unproductive chromosome but the former has retained γ2a, and α, the latter only α. Digestion-circularization (DC)-PCR experiments provide formal proof of DNA recombination between Ca and the intron upstream of Cα. Evidently, the productive chromosome has switched only as far as γ2a, the unproductive all the way to the a constant region gene. The unproductive allele is transcriptionally active as evidenced by the presence of mRNA encoding Cal inappropriately spliced to a cryptic splice site in the downstream intron of DQ52 (eliminated from the productive chromosome). A specific RT-PCR using oligonucleotide primers derived from the upstream initiation site of the la exon and from Cal discloses that T560 cells contain a-germ line mRNA, presumably transcribed from the la-region of the productive chromosome, spliced to Cα. Treatment with LPS stops production of these spliced transcripts suggesting that it may promote either DNA recombination in cells spontaneously transcribing la or a change in splicing such that la sequence is no longer joined to Ca. Verification of the DC-PCR product by sequencing reveals that the T560 and B10.A IgA (Ig2b allotype) hinge is different from the BALB/c IgA (Ig2a allotype) hinge: it has two extra Cys and has eliminated the first Thr, a potential glycosylation site in BALB/c IgA.

End-linked homodimers in fibrinogen Osaka VI with a Bβ-chain extension lead to fragile clot structure

AbstractThe authors have identified a 12-residue carboxyl-terminal extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A mutation that creates AAG encoding Lys at the stop (TAG) codon, thus translating 36 base pairs in the noncoding region of the Bβ gene. The extra Cys residues appear to be involved in 1 or 2 disulfide bonds between 2 adjacent abnormal fibrinogen molecules, forming a fibrinogen homodimer as indicated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Indeed, about half of the fibrinogen molecules exist as end-linked dimers oriented in parallel or with an angle, as observed by transmission electron microscopy. These end-linked dimers may well alter the conformations of D and DD regions on fibrin assembly, leading to increased fiber branching at their sites in the growing protofibrils. By scanning electron microscopy, the Osaka VI fibrin network appears to have a lacelike structure composed of highly branched, thinner fibers than the normal fibrin architecture. Such fibrin networks may be easily damaged to form large pores when fluids are allowed to pass through the gels. The fragility of Osaka VI fibrin clots, further confirmed by permeation and compaction studies, may account for the massive bleeding observed in this patient.

End-linked homodimers in fibrinogen Osaka VI with a Bβ-chain extension lead to fragile clot structure

The authors have identified a 12-residue carboxyl-terminal extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A mutation that creates AAG encoding Lys at the stop (TAG) codon, thus translating 36 base pairs in the noncoding region of the Bβ gene. The extra Cys residues appear to be involved in 1 or 2 disulfide bonds between 2 adjacent abnormal fibrinogen molecules, forming a fibrinogen homodimer as indicated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Indeed, about half of the fibrinogen molecules exist as end-linked dimers oriented in parallel or with an angle, as observed by transmission electron microscopy. These end-linked dimers may well alter the conformations of D and DD regions on fibrin assembly, leading to increased fiber branching at their sites in the growing protofibrils. By scanning electron microscopy, the Osaka VI fibrin network appears to have a lacelike structure composed of highly branched, thinner fibers than the normal fibrin architecture. Such fibrin networks may be easily damaged to form large pores when fluids are allowed to pass through the gels. The fragility of Osaka VI fibrin clots, further confirmed by permeation and compaction studies, may account for the massive bleeding observed in this patient.

Cloning and Modeling of the First Nonmammalian CD4

We have cloned and sequenced the first nonmammalian CD4 cDNA from the chicken using the COS cell expression method. Chicken CD4 contains four extracellular Ig domains that, in analogy to mammalian CD4, are in the order V, C2, V, and C2. The molecule is 24% identical with both human and mouse sequences. The extracellular domains were modeled using human and rat CD4 crystal structures as templates. In the first domain there are two extra Cys residues that are at suitable distance to form an intra-beta-sheet disulfide bridge in addition to the canonical one in the V domain. The region responsible for the interaction with MHC class II is relatively nonconserved in chicken. However, there are positively charged amino acids in the C" region of the N-terminal domain that may mediate the association to the negatively charged residues of the MHC class II beta-chain. Molecular modeling also implies that the membrane-proximal domain mediates dimerization of chicken CD4 in a similar way as it does for human CD4. Furthermore, the cytoplasmic tail is highly conserved, containing the protein tyrosine kinase p56lck recognition site that is preceded by an adjacent di-leucine motif for the internalization of the molecule. Interestingly, there are no Ser residues in the cytoplasmic part, which may explain the slow down-regulation of chicken CD4 after phorbol ester stimulation.