H1 Domains Research Articles

Keratin 1 Gene Mutation Detected in Epidermal Nevus with Epidermolytic Hyperkeratosis

Since 1994, four cases of epidermal nevus with epidermolytic hyperkeratosis (EH) caused by keratin 10 gene mutations have been reported, although no keratin 1 (K1) gene mutation has yet been reported. We detected a K1 gene (KRT1) mutation in epidermal nevus with EH in a 10-year-old Japanese male. The patient showed well-demarcated verrucous, hyperkeratotic plaques mainly on the trunk, covering 15% of the entire body surface. No hyperkeratosis was seen on the palms or soles. He had no family history of skin disorders. His lesional skin showed typical granular degeneration and, ultrastructurally, clumped keratin filaments were observed in the upper epidermis. Direct sequence analysis of genomic DNA extracted from lesional skin revealed a heterozygous 5' donor splice site mutation c.591+2T>A in KRT1. This mutation was not detected in genomic DNA samples from the patient's peripheral blood leukocytes or those of other family members. The identical splice mutation was previously reported in a family with palmoplantar keratoderma and mild ichthyosis, and was demonstrated to result in a 22 amino-acid deletion p.Val175_Lys196del in the H1 and 1A domains of K1. To our knowledge, the present patient is the first reported case of epidermal nevus associated with EH caused by a K1 gene mutation in a mosaic pattern.

A novel H1 domain mutation in the keratin 2 gene in a Japanese family with ichthyosis bullosa of Siemens

Conflicts of interest: none declared. Ichthyosis bullosa of Siemens (IBS) is a rare autosomal dominant skin disorder characterized by blister formation in the upper suprabasal layers of the epidermis. Clinical features of the disease, which include blistering after mechanical trauma, lichenified hyperkeratosis over the flexural areas of the limbs, and superficial peeling of the skin, are similar to those of epidermolytic hyperkeratosis/bullous congenital ichthyosiform erythroderma (EHK/BCIE). This clinical resemblance makes diagnosis difficult. Recent molecular studies have identified mutations in the keratin 2 (K2) gene (KRT2; this new designation is used throughout this report according to the current nomenclature for mammalian keratins1) in patients with IBS, in contrast to the keratin 1 or 10 mutations that have been detected in patients with EHK/BCIE, enabling us to differentiate between IBS and EHK/BCIE.2 Thus far, 29 missense mutations that result in amino acid substitutions in K2 have been identified in patients with IBS; all such mutations reside in the helix initiation or termination motifs of K2. Here, we describe a novel mutation in the H1 region of K2 in a Japanese family with IBS.

Novel mutation (Asp158Val) in H1 domain of keratin 5 gene in a Japanese patient with Köbner‐type epidermolysis bullosa simplex

We describe a 19-year-old Japanese male with Köbner-type epidermolysis bullosa simplex (EBS-KB) with a novel keratin gene mutation. The patient developed blisters on the feet, palms, elbows and knees soon after birth. His father is similarly affected with blistering, but his mother and younger brother are not affected. Histological examination revealed that the primary separation in the blister occurred within the basal cell layer. Sequence analysis demonstrated an A-to-T transition at the second position of codon 158 in the keratin 5 (K5) gene. The amino acid at codon 158 was deduced to have changed from asparagine to valine. We identified a novel mutation (Asp158Val) in the H1 domain of the K5 gene in this Japanese patient with EBS-KB. This is the first gene mutation report of EBS-KB in the H1 domain of the K5 gene.

Structure-based Stability Engineering of the Mouse IgG1 Fab Fragment by Modifying Constant Domains

A semi-rational approach based on structural data was exploited in a search for C H1 and C L domains with improved intrinsic thermodynamic stabilities. Structural and amino acid level comparisons were carried out against known biophysically well-behaving and thermodynamically beneficial scFv and Fab fragments. A number of mutant Fab fragments were constructed by site-directed mutagenesis of regions in the C H1 and C L domains expected to be most sensitive under physical stress conditions. These mutations were located on three sites in the Fab constant domains; a mobile loop in the C H1 domain, residues surrounding the two largest solvated hydrophobic cavities located in the interface of the C H1 and C L domains and the hydrophobic core regions of both C H1 and C L. Expression levels of functional Fab fragments, denaturant-induced unfolding equilibria and circular dichroism spectroscopy were used to evaluate the relative stabilities of the wild-type and the mutant Fab fragments. The highest thermodynamic stability was reached through the mutation strategy, where the hydrophobicity and the packing density of the solvated hydrophobic cavity in the C H1/C L interface was increased by the replacement of the hydrophilic Thr178 in the C L domain by a more hydrophobic residue, valine or isoleucine. The midpoint of the transition curve from native to unfolded states of the protein, measured by fluorescence emission, occurred at concentrations of guanidine hydrochloride of 2.4 M and 2.6 M for the wild-type Fab and the most stable mutants, respectively. Our results illustrate that point mutations targeted to the C H1/C L interface were advantageous for the overall thermodynamic stability of the Fab fragment.

Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small Transmembrane Protein

Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusion-inducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N(exoplasmic)/C(cytoplasmic) topology. This topology results in the translocation of the smallest ectodomain ( approximately 20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of protein-mediated membrane fusion.

Construction of an EGF receptor-mediated histone H1 0 -based gene delivery system

To construct an EGF receptor (EGF-R)-mediated histone H1(0)-based gene delivery system for gene therapy. A recombinant DNA containing histone H1(0), EGF-R ligand, and endosomalytic domains was constructed in a prokaryotic vector and expressed in E. coli. Expression of the beta-galactosidase (beta-gal) gene in the tumor cells and tissues was observed after transduction of the beta-gal gene packaged by purified fusion proteins in vitro and in vivo. As an extension of the research on previously reported chemically synthetic composite polypeptide gene delivery systems, this genetically engineered polypeptide has proved to be capable of targeting the beta-galactosidase (beta-gal) gene into EGF-R-positive cancer cells both in vitro and in vivo. We also studied the time course of beta-gal gene expression in tumor tissues delivered in vivo by this polypeptide vector. At 24 h after administration, expression of the beta-galactosidase gene in tumor reached peak levels. The dosage optimization of administered polyplex was also investigated. The optimal dose of polyplex per mouse was 1 microg DNA packaged by 3 microg of composite polypeptide. The genetically engineered polypeptide based on histone H1(0) is a promising gene delivery system targeting EGF-R.

Lessons from disorders of epidermal differentiation-associated keratins.

A number of diseases have been associated with mutations in genes encoding keratin intermediate filaments. Several of these disorders have skin manifestations, in which histological changes highlight the role of various different keratins in epidermal differentiation. For example, mutations in either K1 or K10 (the major keratin pair expressed in differentiated keratinocytes) usually lead to clumped keratin filaments and cytolysis. Furthermore, the precise nature of the mutation has direct implications for disease phenotype. Specifically, mutations in the H1 and alpha-helical rod domains of K1/K10 result in bullous congenital ichthyosiform erythroderma, underscoring the critical role for this keratin filament domain in maintaining cellular integrity. However, a lysine to isoleucine substitution in the V1 domain of K1 underlies a form of palmoplantar keratoderma, which has different cell biological implications. Keratins are cross-linked into the cornified cell envelopes through this particular lysine residue and the consequences of the mutation lead to changes in keratin-desmosome association and cornified cell morphology, suggesting a role for this keratin subdomain in cornified cell envelope formation. Recently, to extend genotype-phenotype correlation, a frameshift mutation in the V2 region of the K1 tail domain was identified in ichthyosis hystrix (Curth-Macklin type), in which keratin filaments show a characteristic shell-like structure and fail to form proper bundles. In this case, the association of desmosomes with loricrin was also altered, implicating this keratin domain in organizing the intracellular distribution of loricrin during cornification. Collectively, these mutations in K1/K10 provide a fascinating insight into both normal and abnormal processes of epidermal differentiation.

Unassembled Ig Heavy Chains Do Not Cycle from BiP In Vivo but Require Light Chains to Trigger Their Release

Unassembled Ig heavy chains are retained in the ER via the binding of BiP to the C H1 domain, which remains unoxidized. Interestingly, this domain folds rapidly, albeit nonproductively, when heavy chains are released from BiP in vitro with ATP. The in vivo cycling of BiP from heavy chains was monitored using BiP ATPase mutants as kinetic traps. Our data suggest that BiP does not cycle from the C H1 domain of free heavy chains. However, heavy and light chain assembly occurs rapidly and requires the ATP-dependent release of BiP. We propose that BiP's ATPase cycle is stalled or nonproductive when it is bound to free heavy chains. The binding of light chains to the complex reactivates the cycle and releases BiP.

Investigating the Structure of Human RNase H1 by Site-directed Mutagenesis

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.

Recombinant antibodies as carrier proteins for sub-unit vaccines: influence of mode of fusion on protein production and T-cell activation

A major objective in development of vaccines is the design of sub-unit vaccines with the ability to induce strong T-cell responses. For this purpose, T-cell epitopes have been genetically inserted into various carrier proteins. Ig molecules may be especially useful as vehicles for delivery of CD4 + T-cell epitopes to antigen presenting cells (APC). We have previously replaced loop structures between β-strands in the C H1 domain of human IgG3 with a defined 11 amino acids long, MHC class II-restricted T-cell epitope. In this report we have added the same T-cell epitope into loops in the C H1 domain of mouse IgG2b. The following major points can be made: (1) Loops can accommodate an elongation of at least 11 amino acids without disruption of the overall Ig structure and secretion. (2) The recombinant Ig molecules are processed by spleen APC and the epitopes that are released are presented to T-cells. (3) Site of integration influences efficiency of processing and presentation. (4) Elongation of two neighbouring loops reduces Ig secretion. Taken together, our present results indicate that IgG C H1 domains may be engineered to carry T-cell epitopes in loop structures between β-strands, but not all loops may be equally suitable for this purpose.

Functional Cooperation of Two Independent Targeting Domains in Syntaxin 6 Is Required for Its Efficient Localization in thetrans-Golgi Network of 3T3L1 Adipocytes

To identify the targeting domains of syntaxin 6 responsible for its localization to the trans-Golgi network (TGN), we examined the subcellular distribution of enhanced green fluorescent protein (EGFP) epitope-tagged syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncation/deletion mutants in 3T3L1 adipocytes. Expression of EGFP-syntaxin 6 resulted in a perinuclear distribution identical to endogenous syntaxin 6 as determined both by confocal fluorescence microscopy and subcellular fractionation. Furthermore, both the endogenous and the expressed EGFP-syntaxin 6 fusion protein were localized to a brefeldin A-insensitive but okadaic acid-sensitive compartment characteristic of the TGN. In contrast, EGFP-syntaxin 6 constructs lacking the H2 domain were excluded from the TGN and were instead primarily localized to the plasma membrane. Although syntaxin 4 was localized to the plasma membrane, syntaxin 6/syntaxin 4 chimerae and syntaxin 6 truncations containing the H2 domain of syntaxin 6 were predominantly directed to the TGN. Importantly, the syntaxin 6 H2 domain fused to the transmembrane domain of syntaxin 4 was also localized to the TGN, demonstrating that the H2 domain was sufficient to confer TGN localization. In addition to the H2 domain, a tyrosine-based plasma membrane internalization signal (YGRL) was identified between the H1 and H2 domains of syntaxin 6. Deletion of this sequence resulted in the accumulation of the EGFP-syntaxin 6 reporter construct at the plasma membrane. Together, these data demonstrate that syntaxin 6 utilizes two distinct domains to drive its specific subcellular localization to the TGN.

Post-translational modifications of immunoglobulin G: a mouse IgG variant that lacks the entire C H1 domain

In the present study, we characterized the post-translational modifications of a short-chain variant of mouse IgG2a that lacks the entire C H1 domain. The short-chain IgG2a and its proteolytic fragments were subjected to electrosplay ionization- and fast atom bombardment-mass spectrometric analyses. It has been demonstrated that approximately 14% of the heavy chain of the short-chain IgG2a is O-glycosylated with a disaccharide of Gal-GalNAc- at Thr220A in the hinge region, while the O-glycosylation does not occur in its parent IgG2a molecule. Two additional modifications have been detected at the C-termini of both the heavy and light chains of the short-chain IgG2a. Biological significance of the post-translational modifications of the short-chain IgG2a variant is briefly discussed.

Loss of splice consensus signal is responsible for the removal of the entire C H1 domain of the functional camel IGG2A heavy-chain antibodies

The molecular basis for the absence of the C H1 domain in naturally occurring heavy-chain antibodies of the camelids was assessed by determining the entire Camelus dromedarius γ2a heavy-chain constant gene. The organization of the camel γ2a constant heavy-chain gene obtained from a liver genomic library appears to be typical of all other mammalian γ genes sequenced to date. It contains the switch, C H1, hinge, C H2, C H3, M1 and M2 exons. In contrast to the case in mouse and human heavy chain diseases, the camel γ2a gene shows no major structural defect, and its equivalent C H1 exon is intact. However, sequence analysis has revealed that the splicing site, immediately after the C H1 exon, is defective due to point mutations, especially the G +1 to A +1 transversion seems to be detrimental. It is concluded that the loss of the splice consensus signal is responsible for the removal of the entire C H1 domain in camel γ2a heavy-chain immunoglobulins. Additionally, a closer analysis of the hinge exon suggests the possible involvement of transposons in the genetic variation of mammalian C γ hinges.

Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) arises from mutations within the keratin 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocytes cytoskeleton. The majority of these defects are missense mutations in the rod domain, whose locations influence the disease severity. We investigated a large family dominantly affected with the Dowling-Meara form of EBS (EBS-DM). Sequencing of amplified and cloned K5 cDNA from cultured keratinocytes revealed a 66 nucleotide deletion in one allele corresponding to the last 22 amino acid residues encoded by exon 1 (Val164 to Lys185). Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygous G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5. This mutation leads to the use of an exonic GT cryptic donor splice site, located 66 nucleotides upstream from the normal donor splice site of intron 1. The corresponding peptide deletion includes the last five amino acids of the H1 head domain and the first 17 amino acids of the conserved amino terminal end of the 1A rod domain, including the first two heptad repeats and the helix initiation peptide. The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein. This is the first splice site mutation to be reported as a cause of EBS-DM. Owing to the functional importance of the removed region, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.

Control of shortening speed in single guinea-pig taenia coli smooth muscle cells by Ca2+, phosphorylation and caldesmon.

We studied the effect of caldesmon peptides on the regulation of shortening of single guinea-pig taenia coli cells permeabilised with saponin. When contraction was initiated by Ca2+ and MgATP shortening rate at pCa 4.5 was 0.17+/-0.04 cell lengths s-1 and half-maximal rate was at pCa 5.6. Following thiophosphorylation with 1 mM adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) at pCa 4.5 for 10 min, on addition of ATP these cells contracted at of 0.25+/-0.04 cell lengths s-1 independently of pCa. If thiophosphorylated cells were preincubated with H1 (domains 3 and 4 of caldesmon), shortening speed was reduced (ID50=2 microM). Shortening speed was also reduced by 658C (domain 4b) at higher concentrations (ID50=400 microM). H13 (domain 4a), which does not block weak binding but inhibits actin-tropomyosin, inhibited cell shortening (ID50=6 microM). H2, which blocks weak binding but does not inhibit actin-tropomyosin, did not inhibit shortening. Western blots of the cells showed that the peptides were tightly bound within the cell but the native caldesmon was not displaced. These results indicate that exogenous caldesmon peptides added to smooth muscle cells may be incorporated into the thin filaments and produce effects on shortening, as expected if it were involved in tropomyosin-dependent inhibition of the actin filament in the cell.

Novel K5 and K14 Mutations in German Patients with the Weber–Cockayne Variant of Epidermolysis Bullosa Simplex

We report novel keratin 5 and 14 gene mutations in four unrelated German families with the localized subtype of the dominantly inherited blistering disease epidermolysis bullosa simplex Weber-Cockayne (MIM# 131800). The mutations are located in the keratin 14 L12 linker region (D273G), the keratin 5 L12 linker (M327K and D328H), and the H1 domain of keratin 5 (P156L). These mutations add to those previously reported and provide further evidence of phenotype-genotype correlations in epidermolysis bullosa simplex subtypes. The above mutations in mildly affected patients underline the relevance of the keratin linker regions for the epidermolysis bullosa simplex Weber-Cockayne phenotype and keratin filament integrity. In addition, they confirm that the gene segments encoding the linker regions represent hotspots for mutations.

The first constant domain (C H1 and C L) of an antibody used as heterodimerization domain for bispecific miniantibodies

Bispecific miniantibodies were constructed by genetically fusing the C H1 domain of an IgG1 to the C-terminus of a single-chain Fv fragment (scFv-425), specific for the EGF receptor, and fusing the C L domain of a kappa light chain to the C-terminus of a scFv specific for CD2 (scFv-M1). An efficient dicistronic gene arrangement for functional expression in Escherichia coli was constructed. Immunoblots demonstrated correct domain assembly and the formation of the natural C H1-C L disulfide bridge. Gel filtration confirmed the correct size, sandwich ELISAs demonstrated bispecific functionality, and SPR biosensor measurements determined binding to EGF-R in comparison to bivalent constructs. Bispecific anti-EGF-R/anti-CD2 miniantibodies are candidates for the immunotherapy of cancer.

Polypeptide Chain Folding in the Hydrophobic Core of Hamster Scrapie Prion: Analysis by X-Ray Diffraction

Conversion of the noninfectious, cellular form of the scrapie prion (PrPC) to the infectious form (PrPSc) is thought to be driven by an α-helical to β-sheet conformational transition. The N-truncated polypeptide PrP27–30, which encompasses residues 90–231 of PrPScand from which the truncated peptide is derived by limited proteolysis, assembles into amyloid rods that are rich in the β-sheet conformation. The N-terminal half of PrP27–30, which includes residues 90–145 of PrP (SHa90–145) and contains the two putative α-helical domains H1 (PrP109–122) and H2 (PrP129–141), appears to be particularly crucial in the α → β conversion. To assess their role in this conformational transition, we have analyzed in detail X-ray diffraction patterns from the prion-related peptides A8A (PrP113–120), H1, and SHa90–145. We used iterative Fourier synthesis with β-silk as an initial model for assigning phases. For H1, the lyophilized and acetonitrile-solubilized/dehydrated specimens gave two different electron density maps. The former showed that the β-sheets were composed of small side chains as in A8A. The latter showed two types of β-sheets having smaller and larger side chains, suggesting a turn. Such a turn was not observed in the lyophilized H1, indicating that the internal turn in H1 depends on the physical–chemical environment. In SHa90–145, the β-chains are assembled in ≈40 Å-wide crystal domains (termed β-crystallites), and the electron density maps of these crystallites showed evidence for turns within both the H1 and H2 domains. The molecular folding of H1–H2 is compared here with the recent NMR solution structure of recombinant hamster prion, and the effect of pH on the conformational change is discussed. The most compact structure based on the X-ray diffraction analysis showed that the N-terminal, smaller residues of H2 fold back and are hydrogen-bonded with the C-terminal, smaller residues of H1. Similar folding is observed in the NMR solution structure. Comparison of the NMR structures at different pH with the X-ray diffraction results suggests that histidine and lysine residues in the N-terminal sequence of PrP may figure in the α → β structure transition of PrP.

X-ray diffraction analysis of scrapie prion: intermediate and folded structures in a peptide containing two putative α-helices

Small proteinaceous infectious particles called prions cause certain neuro-degenerative diseases in human and animals. Limited proteolysis of infectious scrapie prions PrPSc yields an N-truncated polypeptide termed PrP 27-30, which encompasses residues 90 to 231 of PrPSc and which assembles into 100 to 200 Å wide amyloid rods. It has been hypothesized that the infectious prion is converted from its non-infectious cellular form (PrPC) by means of an α-helical to β-sheet conformational change. Secondary structure analysis, computer modeling, and structural biophysics methods support this hypothesis. Residues 90 to 145 of PrP, which contain two putative α-helical domains H1 and H2, may be of particular relevance to the disease pathogenesis, as C-terminal truncation at residue 145 was found in a patient with an inherited prion disease. Moreover, our recent X-ray diffraction analysis suggests that the peptide consisting of these residues (designated SHa 90-145) closely models the amyloidogenic β-sheet core of PrP. In the current study, we have analyzed in detail the X-ray diffraction patterns of SHa 90-145. Two samples were examined: one that was dehydrated under ambient conditions whilst in an external magnetic field (to induce fibril orientation), and another that was sealed after partial drying. The dried, magnetically oriented sample showed a cross-β diffraction pattern in which the fiber axis (rotation axis) was parallel to the H-bonding direction of the β-sheets. The major wide-angle peaks indicate the presence of ∼40 Å wide β-crystallites, which constitute the protofilament. Each crystallite is composed of several orthogonal unit cells, normal to the fiber (a-axis) direction, having lattice constants a=9.69 Å, b=6.54 Å, and c=18.06 Å. Electron density maps were calculated by iterative Fourier synthesis using β-silk as an initial phase model. The distribution of density indicated that there were two types of β-sheet, suggesting that larger and smaller side-chains localized to different sheets. This would arise from folding of the polypeptide in which there are turns in the middle of both the H1 and H2 domains. A monoclinic macrolattice, with a=9.61 Å, b=c=52.99 Å and α=114.6°, was found to index all the reflections, including those in the low-angle region. This suggests that the β-crystallites are nearly hexagonally packed. To account for the ∼100 Å wide fibers visualized by negative staining in the electron microscope, the β-crystallites would be arranged in 4-mers. The partially dried sample showed a sharp 4.7 Å reflection (from H-bonding) and five broad peaks superimposed on monotonically decreasing diffuse scattering. This solution-like scattering was modeled by an anisometric rectangle with a thickness comparable to a singe β-chain. The structure, which occurred during dehydration, could be a transient in the α-helical to β-sheet conversion, suggesting that formation of hydrogen bonding precedes the inter-sheet interaction and assembly into the amyloid of scrapie prion.

Coordination of immunoglobulin chain folding and immunoglobulin chain assembly is essential for the formation of functional IgG

The first constant domain (C H1) of immunoglobulin heavy (H) chains is essential for BiP-mediated retention of unassembled H chains in the endoplasmic reticulum (ER). Here, we demonstrated that both wild-type and a mutant γ chain lacking the CHI domain bind BiP when they are reduced In vivo. However, only oxidized mutant H chain dimers are released from BiP interaction, whereas oxidized wild-type γ chain dimers still bind BiP. In light (L) chain-producing cells, some of the mutant H chains accumulate with L chains in ER-derived vesicles and some are secreted as IgG. Furthermore, only half of the secreted antibodies bind antigen. We found the same with a mutant γ chain, in which the C 3 domain was replaced by a C H1 domain. Therefore, we propose that BiP Interaction with incompletely folded C H1 domains Is required to mediate correct assembly of H and L chains.