Interchain Disulphide Bonds Research Articles

Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro.

Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.

Novel Small Molecule Inhibitors That Prevent the Neuroparalysis of Tetanus Neurotoxin.

Tetanus neurotoxin (TeNT) is a protein exotoxin produced by Clostridium tetani that causes the deadly spastic neuroparalysis of tetanus. It consists of a metalloprotease light chain and of a heavy chain linked via a disulphide bond. TeNT binds to the neuromuscular junction (NMJ) and it is retro-axonally transported into vesicular compartments to the spinal cord, where it is released and taken up by inhibitory interneuron. Therein, the catalytic subunit is translocated into the cytoplasm where it cleaves its target protein VAMP-1/2 with consequent blockage of the release of inhibitory neurotransmitters. Vaccination with formaldehyde inactivated TeNT prevents the disease, but tetanus is still present in countries where vaccination coverage is partial. Here, we show that small molecule inhibitors interfering with TeNT trafficking or with the reduction of the interchain disulphide bond block the activity of the toxin in neuronal cultures and attenuate tetanus symptoms in vivo. These findings are relevant for the development of therapeutics against tetanus based on the inhibition of toxin molecules that are being retro-transported to or are already within the spinal cord and are, thus, not accessible to anti-TeNT immunoglobulins.

PO-418 ImmTAC® molecules: cancer treatment through soluble, high affinity T cell receptors

Introduction Tumour associated antigen (TAA) peptides presented on cancer cells in complex with human leukocyte antigen (HLA) molecules provide a marker for targeting by cytotoxic T cells. The ability of T cells to identify aberrant peptides presented on malignant cells is achieved through a membrane-anchored a/b heterodimer called the T cell receptor (TCR). Whilst patients frequently mount anti-tumour T cell responses, often these are insufficient to clear the tumour. This can be attributed to the low level expression of tumour peptides on cancer cells and to the low binding affinity of TCRs targeting cancer antigens. To overcome the target recognition limitations of the naturally occurring T cell pool, we have developed a new class of soluble bi-specific molecules called ImmTAC (immune mobilising monoclonal TCRs against cancer) which combine a tumour targeting domain, an affinity-engineered TCR and a T cell-activating effector function, an anti-CD3 scFv. Material and methods T cell clones are isolated from human blood, which harbour wild-type TCRs recognising peptides derived from TAAs. The TCR α- and β- chains are sequenced and a stabilising non-native inter-chain disulphide bond is introduced. The TCR chains are expressed as inclusion bodies in Escherichia coli and are refolded in vitro to produce soluble and stable protein. The binding affinity is determined by surface plasmon resonance (SPR) and is typically in the range ~1–400 mM with half-lives of 1–10 s. For effective tumour targeting, the TCR is engineered using phage display to enhance affinity for cognate antigen by a million-fold or greater. To generate an ImmTAC molecule, the soluble TCR is fused to a humanised anti-CD3 scFv effector function and potency and specificity is assessed in vitro against HLA-relevant antigen positive cancer cell lines and cultured primary cells isolated from normal human tissues. Results and discussions Our lead clinical candidate, IMCgp100, targets the HLA-A*02/gp100280–288 epitope expressed at the surface of melanoma cells. Using directed molecular evolution and phage display targeting the complementarity-determining regions (CDRs) we have produced a bispecific reagent with picomolar binding affinity and a binding half-life of several hours. IMCgp100 has entered pivotal monotherapy trials for the treatment of patients with metastatic uveal melanoma. Conclusion The ImmTAC technology platform is a new class of bi-specific biologic based on T cell receptors. IMCgp100 is the first ImmTAC in clinical trials to treat advanced melanoma.

Wheat glutenin: the “tail” of the 1By protein subunits

Gluten-forming storage proteins play a major role in the viscoelastic properties of wheat dough through the formation of a continuous proteinaceous network. The high-molecular-weight glutenin subunits represent a functionally important subgroup of gluten proteins by promoting the formation of large glutenin polymers through interchain disulphide bonds between glutenin subunits. Here, we present evidences that y-type glutenin subunits encoded at the Glu-B1 locus are prone to proteolytic processing at the C-terminus tail, leading to the loss of the unique cysteine residue present at the C-terminal domain. Results obtained by intact mass measurement and immunochemistry for each proteoform indicate that the proteolytic cleavage appears to occur at the carboxyl-side of two conserved asparagine residues at the C-terminal domain start. Hence, we hypothesize that the responsible enzymes are a class of cysteine endopeptidases - asparaginyl endopeptidases - described in post-translational processing of other storage proteins in wheat.Biological significanceThe reported study provides new insights into wheat storage protein maturation. In view of the importance of gluten proteins on dough viscoelastic properties and end-product quality, the reported C-terminal domain cleavage of high-molecular-weight glutenin subunits is of particular interest, since this domain possesses a unique conserved cysteine residue which is assumed to participate in gluten polymerization.

The thioredoxin reductase – Thioredoxin redox system cleaves the interchain disulphide bond of botulinum neurotoxins on the cytosolic surface of synaptic vesicles

Botulinum neurotoxins (BoNTs) are Janus toxins, as they are at the same time the most deadly substances known and one of the safest drugs used in human therapy. They specifically block neurotransmission at peripheral nerves through the proteolysis of SNARE proteins, i.e. the essential proteins which are the core of the neuroexocytosis machinery. Even if BoNTs are traditionally known as seven main serotypes, their actual number is much higher as each serotype exists in many different subtypes, with individual biological properties and little antigenic relations. Since BoNTs can be used as biological weapons, and the only currently available therapy is based on immunological approaches, the existence of so many different subtypes is a major safety problem. Nevertheless, all BoNT isoforms are structurally similar and intoxicate peripheral nerve endings via a conserved mechanism. They consist of two chains linked by a unique disulphide bond which must be reduced to enable their toxicity. We found that thioredoxin 1 and its reductase compose the cell redox system responsible for this reduction, and its inhibition via specific chemicals significantly reduces BoNTs activity, in vitro as well as in vivo. Such molecules can be considered as lead compounds for the development of pan-inhibitors.

Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins

To maintain proteostasis in the endoplasmic reticulum (ER), terminally misfolded secretory proteins must be recognized, partially unfolded, and dislocated to the cytosol for proteasomal destruction, in a complex process called ER-associated degradation (ERAD). Dislocation implies reduction of inter-chain disulphide bonds. When in its reduced form, protein disulphide isomerase (PDI) can act not only as a reductase but also as an unfoldase, preparing substrates for dislocation. PDI oxidation by Ero1 favours substrate release and transport across the ER membrane. Here we addressed the redox dependency of ERAD and found that DTT stimulates the dislocation of proteins with DTT-resistant disulphide bonds (i.e., orphan Ig-μ chains) but stabilizes a ribophorin mutant (Ri332) devoid of them. DTT promotes the association of Ri332, but not of Ig-µ, with PDI. This discrepancy may suggest that disulphide bonds in cargo proteins can be utilized to oxidize PDI, hence facilitating substrate detachment and degradation also in the absence of Ero1. Accordingly, Ero1 silencing retards Ri332 degradation, but has little if any effect on Ig-µ. Thus, some disulphides can increase the stability and simultaneously favour quality control of secretory proteins.

Complementary DNA sequences of the constant regions of T‐cell antigen receptors α, β and γ in mandarin fish, Siniperca chuatsi Basilewsky, and their transcriptional changes after stimulation with Flavobacterium columnare

In this study, the constant-region genes (Cα, Cβ and Cγ) that encode the T-cell antigen receptor (TCR) α, β and γ chains were cloned from mandarin fish, Siniperca chuatsi Basilewsky, an important freshwater fish species in China. The complementary DNA sequences of Cα, Cβ and Cγ were 843, 716 and 906base pairs (bp) in length and had a 465-, 289- and 360-bp 3' untranslated region, encoding 125, 142 and 182 amino acids, respectively. The amino-acid sequences of the constant regions of mandarin fish TCR α, β and γ chains (encoded by Cα, Cβ and Cγ, respectively) were most similar to those of their teleost counterparts, showing 60% similarity with pufferfish, 48% similarity with Atlantic salmon and 57% similarity with flounder, respectively. The phylogenetic analysis revealed that the mandarin fish Cα, Cβ and Cγ were clustered, respectively, with their vertebrate counterparts. The mandarin fish Cα, Cβ and Cγ could also be separated into four domains: immunoglobulin; connecting peptide (CP); transmembrane (TM); and cytoplasmic tail. Several conserved features in mammalian TCRs were also found in those of mandarin fish, such as a conserved cysteine residue in the CP domain of Cα, necessary for creating an interchain disulphide bond with the TCR β chain, and a conserved antigen receptor TM motif in Cα and Cβ. Meanwhile, transcripts of Cα, Cβ and Cγ were detectable in all examined organs, with a stronger signal observed in lymphoid organs. In addition, the temporal transcriptional changes for Cα and Cγ were investigated, 1, 2, 3, 4, 5, 6 and 8weeks after stimulation with Flavobacterium columnare, in head kidney, spleen, blood, thymus, gill and intestine, using real-time polymerase chain reaction. The results demonstrated stimulation-dependent up-regulations in almost all tissues examined, which indicates that T cells may play important roles in preventing mandarin fish from bacterial invasion. In particular, apart from thymus, T cells were distributed mainly in gill and intestine, where striking up-regulation of Cγ was also observed. These results will facilitate functional studies of teleost TCRs and T cells.

Transformation of common wheat (Triticum aestivum L.) with avenin-like b gene improves flour mixing properties.

Avenin-like b proteins may contribute to the viscoelastic properties of wheat dough via inter-chain disulphide bonds, due to their rich cysteine residues. In order to clarify the effect of the avenin-like b proteins on the functional properties of wheat flour, the functional and biochemical properties of wheat flour were analyzed in three transgenic wheat lines overexpressing the avenin-like b gene using the sodium dodecyl sulfate sedimentation (SDSS) test, Mixograph and size exclusion-high performance liquid chromatography (SE-HPLC) analysis. The results of the SDSS test and Mixograph analysis demonstrated that the overexpression of avenin-like b proteins in transgenic lines led to significantly increased SDSS volume and improved flour mixing properties. The results of SE-HPLC analysis of the gluten proteins in wheat flour demonstrated that the improvement in transgenic line flour properties was associated with the increased proportion of large polymeric proteins due to the incorporation of overexpressed avenin-like b proteins into the glutenin polymers. These results could help to understand the influence and mechanism of avenin-like b proteins on the functional properties of wheat flour.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-013-9913-1) contains supplementary material, which is available to authorized users.

Overexpression of avenin-like b proteins in bread wheat (Triticum aestivum L.) improves dough mixing properties by their incorporation into glutenin polymers.

Avenin-like b proteins are a small family of wheat storage proteins, each containing 18 or 19 cysteine residues. The role of these proteins, with high numbers of cysteine residues, in determining the functional properties of wheat flour is unclear. In the present study, two transgenic lines of the bread wheat overexpressing avenin-like b gene were generated to investigate the effects of Avenin-like b proteins on dough mixing properties. Sodium dodecyl sulfate sedimentation (SDSS) test and Mixograph analysis of these lines demonstrated that overexpression of Avenin-like b proteins in both transgenic wheat lines significantly increased SDSS volume and improved dough elasticity, mixing tolerance and resistance to extension. These changes were associated with the increased proportion of polymeric proteins due to the incorporation of overexpressed Avenin-like b proteins into the glutenin polymers. The results of this study were critical to confirm the hypothesis that Avenin-like b proteins could be integrated into glutenin polymers by inter-chain disulphide bonds, which could help understand the mechanism behind strengthening wheat dough strength.

An engineered HIV-1 gp41 trimeric coiled coil with increased stability and anti-HIV-1 activity: implication for developing anti-HIV microbicides

We previously constructed a trimeric coiled coil, N28Fd, based on the N-heptad repeat (NHR) sequence of HIV-1 gp41, as a promising HIV-1 entry inhibitor. Here, we attempted to engineer a stabilized trimeric coiled coil, ccN28Fd, by adding interchain disulphide bonds at the N terminus of N28Fd to improve its biophysical properties and anti-HIV-1 efficacy. Molecular biology techniques were applied to engineer ccN28Fd. Circular dichroism and sedimentation velocity analysis were used to determine its secondary structure and thermostability and polymeric states, respectively. The anti-HIV-1 activity was assessed by p24 or luciferase expression. Its cytotoxicity was evaluated by XTT assay. At low pH, ccN28Fd and N28Fd were in trimeric and monomeric conformation, respectively. ccN28Fd showed higher thermostability and much stronger antiviral activity against HIV-1 IIIB (X4) and Bal (R5) strains than N28Fd. ccN28Fd was effective in inhibiting infection by a broad spectrum of primary HIV-1 isolates and enfuvirtide-resistant strains and blocking cell-to-cell HIV-1 transmission. A combination of ccN28Fd with tenofovir, a nucleoside reverse transcriptase inhibitor-based microbicide, exhibited potent synergistic anti-HIV-1 activity. ccN28Fd was highly resistant to digestion by proteinase K at pH 7.2 and pepsin at pH 1.5, and its anti-HIV-1 activity was not significantly affected by the presence of hydroxyethyl cellulose gel, seminal fluid or vaginal fluid simulant. It possessed no significant in vitro cytotoxicity. The engineered ccN28Fd maintains high stability in a low pH environment and exhibits potent and broad anti-HIV-1 activity, suggesting good potential for development as an effective and safe vaginal microbicide to prevent HIV sexual transmission.

Towards a universal disulphide stabilised single chain Fv format: importance of interchain disulphide bond location and vL-vH orientation

Engineered introduction of interface interchain disulphide bonds is perceived to be a simple method to increase the stability of single chain Fv (scFv). Six disulphide bond locations have been cited within the literature but the potential for the broad use of each has not been examined. Five of these disulphide bond locations were introduced into one scFv in order to compare their relative effects on expression, thermal stability, percent monomer formation and retention of antigen binding. The disulphide bond position vH44-vL100 was observed to enable the most favourable balance of biophysical properties. The vH44-vL100 disulphide bond was introduced into five additional scFv in both vL-vH and vH-vL orientations in order to investigate its general applicability. Data are presented to show the relative influence of scFv sequence, v-region organisation and interchain disulphide bond on expression yield, thermal stability and percent monomer. Introduction of the vH44-vL100 disulphide bond typically resulted in no or little increase in thermal stability and no change in percent monomer but did confer the benefit of permanently fixing monomer:dimer ratios during purification and analysis.

Molecular insight into IgE-mediated reactions to sesame ( Sesamum indicum L.) seed proteins

Food allergy is becoming a major public health concern in recent times. Several sesame seed allergenic proteins have been identified. However, sensitization toward these proteins does not follow a common and unique pattern of clinical reactivity, as shown by the differential geographic recognition of single proteins. To evaluate the sensitization profiles of 18 Italian individuals who experienced clinical symptoms after sesame seed consumption, including 4 anaphylactic reactions. Using an in vitro approach, we adopted a 2-dimensional electrophoretic technique combined with immunoblotting analyses by using sera from 18 Italian sesame-allergic patients. We showed the prevalent and almost exclusive reactivity of the sesame 11S globulin. We shed light on the active role of the basic subunit of this globulin family. The limited accessibility of this polypeptide chain, unless the interchain disulphide bonds are cleaved, may be one of the reasons for its structural/functional stability and, thus, great potential for induction of IgE reactivity. These results confirmed previous findings on the reactivity of the basic subunit of 11S globulin in various legume species. Moreover, this experimental approach proved to be useful for the noninvasive screening of specific reactivities in sensitized patients.

Isolation and characterization of Avenin-like protein type-B from durum wheat

Proteomic techniques were applied for the investigation of “avenin-like protein type-B” in tetraploid wheat. Protein was extracted from endosperm, according to a classical chromatographic procedure and its identity confirmed by liquid chromatography-tandem mass spectrometry analysis. The characterization of avenin-like protein type-B was in good agreement with the gene-derived sequence, with the exception of glutamine as N-terminus. The presence in the glutenin extract and the high number of cysteine residues suggested that avenin-like protein type-B is integrated into gluten polymers via inter-chain disulphide bonds. This raises the question of whether the protein could play a role in determining the functional properties of gluten.

Functional hypothesis on miraculin’ sweetness by a molecular dynamics approach

Miraculin differs from other sweet-tasting proteins because it is a taste-modifier having the unusual property of modifying sourness into sweetness. Its dimer is covalently linked by an inter-chain disulphide bond, and shows its taste-modifying activity at acidic pH, with maximum at pH 3.0, while it is flat at neutral pH. Previous studies suggested the importance of two histidine residues for the taste-modifying activity of miraculin. In this work, we have conducted molecular dynamics simulations on wild type miraculin and on three mutated dimers (H29A, H59A and H29A/H59A) both at neutral and acidic pH to investigate the structural and functional role of these two His residues. Our results suggested that at acidic pH the presence of two charged His at the interface induced a structural rearrangement of the two monomers, thus leading to their relative opening and the following adaptation of their conformation to the receptor surface. On the other hand the simulations on three mutants showed that the mutated dimers had a closed form, and highlighted the important role of H29 in stabilizing/destabilizing the dimer arrangement and also a cooperative effect of the two histidines.

Identification and characterization of nCLP2, a novel C1q family protein expressed in the central nervous system

The C1q family is characterized by the C-terminally conserved globular C1q (gC1q) domain. Although more than 30 C1q family proteins have been identified in mammals, many of them remain ill-defined with respect to their molecular and biological properties. Here, we report on a novel C1q family protein specifically expressed in the central nervous system (CNS), which we designated neural C1q-like protein (nCLP) 2. nCLP2 was secreted as disulphide-bonded multimers comprising trimeric units. The multimers were stabilized by interchain disulphide bonds involving the cysteine residues in the N-terminal variable region and the C-terminal gC1q domain. The expression of nCLP2 was restricted to several brain regions and retina, including regions associated with memory formation (i.e. hippocampus, entorhinal cortex, anterodorsal thalamic nucleus). Immunoelectron microscopy revealed that nCLP2 was localized in the mossy fibre axons of hippocampal granule cells and their synaptic boutons and clefts, implying that nCLP2 was anterogradely transported in mossy fibres and secreted from the presynaptic termini. These results suggest that nCLP2 plays roles in synaptic function and maintenance in the CNS.

Effect of chitosan and thiolated chitosan coating on the inhibition behaviour of PIBCA nanoparticles against intestinal metallopeptidases

Surface modified nanoparticles composed of poly(isobutylcyanoacrylate) (PIBCA) cores surrounded by a chitosan and thiolated chitosan gel layer were prepared and characterized in previous works. The presence of such biopolymers on the nanoparticle surface conferred those nanosystems interesting characteristics that might partially overcome the gastrointestinal enzymatic barrier, improving the oral administration of pharmacologically active peptides. In the present work, the antiprotease behaviour of this family of core–shell nanoparticles was in vitro tested against two model metallopeptidases present in the gastrointestinal tract (GIT): Carboxypeptidase A -CP A- (luminal protease) and Leucine Aminopeptidase M -LAP M- (membrane protease). As previous step, the zinc-binding capacity of these nanoparticles was evaluated. Interestingly, an improvement of both the zinc-binding capacity and the antiprotease effect of chitosan was observed when the biopolymers (chitosan and thiolated chitosan) were used as coating component of the core–shell nanoparticles, in comparison with their behaviour in solution, thanks to the different biopolymer chains rearrangement. The presence of amino, hydroxyl and thiol groups on the nanoparticle surface promoted zinc binding and hence the inhibition of the metallopeptidases analysed. On the contrary, the occurrence of a cross-linked structure in the gel layer surrounding the PIBCA cores of thiolated formulations, due to the formation of interchain and intrachain disulphide bonds, partially limited the inhibition of the proteases. The low accessibility of cations to the active groups of the cross-linked polymeric shell was postulated as a possible explanation of this behaviour. Results obtained in this work make this family of surface-modified nanocarriers promising candidates for the successfull administration of pharmacologically active peptides and proteins by the oral route.

Collagens at a glance

Collagens are a large family of triple helical proteins that are widespread throughout the body and are important for a broad range of functions, including tissue scaffolding, cell adhesion, cell migration, cancer, angiogenesis, tissue morphogenesis and tissue repair. Collagen is best known as the

Alternative antibody Fab′ fragment PEGylation strategies: combination of strong reducing agents, disruption of the interchain disulphide bond and disulphide engineering

Antigen-binding fragments (Fab') of antibodies can be site specifically PEGylated at thiols using cysteine reactive PEG-maleimide conjugates. For therapeutic Fab'-PEG, conjugation with 40 kDa of PEG at a single hinge cysteine has been found to confer appropriate pharmacokinetic properties to enable infrequent dosing. Previous methods have activated the hinge cysteine using mildly reducing conditions in order to retain an intact interchain disulphide. We demonstrate that the final Fab-PEG product does not need to retain the interchain disulphide and also therefore that strongly reducing conditions can be used. This alternative approach results in PEGylation efficiencies of 88 and 94% for human and murine Fab, respectively. It also enables accurate and efficient site-specific multi-PEGylation. The use of the non-thiol reductant tris(2-carboxyethyl) phosphine combined with protein engineering enables us to demonstrate the mono-, di- and tri-PEGylation of Fab fragments with a range of PEG size. We present evidence that PEGylated and unPEGylated Fab' molecules that lack an interchain disulphide bond retain very high levels of chemical and thermal stability and normal performance in PK and efficacy models.

Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei

A natural lectin from the serum of the shrimp Litopenaeus vannamei was purified to homogeneity by a single-step affinity chromatography using fetuin-coupled agarose. The purified serum lectin (named LVL) showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC, chicken RBC and its haemagglutinating (HA) activity was specifically dependent on Ca 2+ and reversibly sensitive to EDTA. LVL inactive form had a molecular mass estimate of 172 kDa and was composed of two non-identical subunits (32 and 38 kDa) cross-linked by interchain disulphide bonds. Significant LVL activity was observed between pH 7 and 11. In HA-inhibition assays performed with several carbohydrates and glycoproteins, LVL showed a distinct and unique specificity for GalNAc/GluNAc/NeuAc which had an acetyl group, while glycoproteins fetuin and bovine submaxillary mucin (BSM) had sialic acid. Moreover, this agglutinin appeared to recognise the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of L. vannamei lectin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections.

Chemistry of gluten proteins

Gluten proteins play a key role in determining the unique baking quality of wheat by conferring water absorption capacity, cohesivity, viscosity and elasticity on dough. Gluten proteins can be divided into two main fractions according to their solubility in aqueous alcohols: the soluble gliadins and the insoluble glutenins. Both fractions consist of numerous, partially closely related protein components characterized by high glutamine and proline contents. Gliadins are mainly monomeric proteins with molecular weights (MWs) around 28,000–55,000 and can be classified according to their different primary structures into the α/ β-, γ- and ω-type. Disulphide bonds are either absent or present as intrachain crosslinks. The glutenin fraction comprises aggregated proteins linked by interchain disulphide bonds; they have a varying size ranging from about 500,000 to more than 10 million. After reduction of disulphide bonds, the resulting glutenin subunits show a solubility in aqueous alcohols similar to gliadins. Based on primary structure, glutenin subunits have been divided into the high-molecular-weight (HMW) subunits (MW=67,000–88,000) and low-molecular-weight (LMW) subunits (MW=32,000–35,000). Each gluten protein type consists or two or three different structural domains; one of them contains unique repetitive sequences rich in glutamine and proline. Native glutenins are composed of a backbone formed by HMW subunit polymers and of LMW subunit polymers branched off from HMW subunits. Non-covalent bonds such as hydrogen bonds, ionic bonds and hydrophobic bonds are important for the aggregation of gliadins and glutenins and implicate structure and physical properties of dough.