Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule.

Recombinant monoclonal antibodies undergo extensive posttranslational modifications. In this article, we characterize major modifications, separated by cation exchange chromatography, on an immunoglobulin G1 (IgG1) monoclonal antibody (mAb). We found that N-terminal cyclization of glutamine residues to pyroglutamate on the light and heavy chains are the major isoforms resolved during cation exchange chromatography. However, using CEX, we also separated and identified isoforms with unpaired cysteine residues in the V(H) domain of the molecule (Cys22-Cys96). Omalizumab, a therapeutic anti-IgE antibody, has unpaired cysteine residues in the V(H) domain between Cys22 and Cys96, and the Fab fragment, containing the unpaired cysteine residues, is reported to have reduced potency. Dynamic interchain disulfide rearrangement, with slow kinetics, was recently reported to take place in serum for an IgG2 molecule and resulted in predictable mature isoforms. Analytical evaluation of our mAb, after recovery from serum, revealed that the unpaired intrachain cysteine residues (Cys22-Cys96) reformed their disulfide bond. The significance of this study is that correct pairing occurred rapidly, and we speculate that thiol molecules such as cysteine, homocysteine, and glutathione in serum provide an environment, outside the endoplasmic reticulum, for correct linkage.

The unfolding of substrates and ubiquitin-independent protein degradation by proteasomes.

26S proteasomes are composed of a 20S proteolytic core and two ATPase-containing 19S regulatory particles. To clarify the role of these ATPases in proteolysis, we studied the PAN complex, the archaeal homolog of the 19S ATPases. When ATP is present, PAN stimulates protein degradation by archaeal 20S proteasomes. PAN is a molecular chaperone that catalyzes the ATP-dependent unfolding of globular proteins. If 20S proteasomes are present, this unfoldase activity is linked to degradation. Thus PAN, and presumably the 26S ATPases, unfold substrates and facilitate their entry into the 20S particle. 26S proteasomes preferentially degrade ubiquitinated proteins. However, we found that calmodulin (CaM) and troponin C are degraded by 26S proteasomes without ubiquitination. Ca(2+)-free native CaM and in vitro 'aged' CaM are degraded faster than the Ca(2+)-bound form. Ubiquitination of CaM does not enhance its degradation. Degradation of ovalbumin normally requires ubiquitination, but can occur without ubiquitination if ovalbumin is denatured. The degradation of these proteins still requires ATP and the 19S particle. Thus, ubiquitin-independent degradation by 26S proteasomes may be more important than generally assumed.

Ca2+-free calmodulin and calmodulin damaged by in vitro aging are selectively degraded by 26 S proteasomes without ubiquitination.

The ubiquitin-proteasome pathway is believed to selectively degrade post-synthetically damaged proteins in eukaryotic cells. To study this process we used calmodulin (CaM) as a substrate because of its importance in cell regulation and because it acquires isoaspartyl residues in its Ca(2+)-binding regions both in vivo and after in vitro "aging" (incubation for 2 weeks without Ca(2+)). When microinjected into Xenopus oocytes, in vitro aged CaM was degraded much faster than native CaM by a proteasome-dependent process. Similarly, in HeLa cell extracts aged CaM was degraded at a higher rate, even though it was not conjugated to ubiquitin more rapidly than the native species. Ca(2+) stimulated the ubiquitination of both species, but inhibited their degradation. Thus, for CaM, ubiquitination and proteolysis appear to be dissociated. Accordingly, purified muscle 26 S proteasomes could degrade aged CaM and native Ca(2+)-free (apo) CaM without ubiquitination. Addition of Ca(2+) dramatically reduced degradation of the native molecules but only slightly reduced the breakdown of the aged species. Thus, upon Ca(2+) binding, native CaM assumes a non-degradable conformation, which most of the age-damaged species cannot assume. Thus, flexible conformations, as may arise from age-induced damage or the absence of ligands, can promote degradation directly by the proteasome without ubiquitination.

Transglutaminase crosslinking and structural studies of the human small proline rich 3 protein.

The cell envelope (CE) is a vital structure for barrier function in terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including SPR3 in certain specialized epithelia normally subjected to mechanical trauma. We have expressed recombinant human SPR3 in order to study its cross-linking properties. It serves as a complete substrate for, and is cross-linked at similar efficiencies by, the three enzymes (TGases 1, 2 and 3) that are widely expressed in many epithelia. Multiple adjacent glutamines (4, 5, 16, 17, 18, 19 and 167) and lysines (6, 21, 164, 166 and 168) of only head and tail domain sequences are used for cross-linking. However, each enzyme preferentially uses certain residues on the head domain. Moreover, our in vitro data suggest a defined temporal order of cross-linking of SPR3 in vivo: It is first cross-linked by TGase 3 into short intra- and inter-chain oligomers which are later further cross-linked to the CE by TGase 1. To investigate the absence of cross-linking in the central domain (e.g. lysine in position 2 of each of the 16 repeats) we performed structural studies on recombinant SPR3 and on a synthetic peptide containing three repeats of the central domain. 2D H-1 NMR spectroscopy, TOCSY and ROESY, shows strong and medium intensity NOEs connectivities along the amino acid sequence with one weak long range NOE contact between Thr and Cys of subsequent repeats. Distance geometry computation on the basis of intensities of NOEs found generated 50 compatible structures grouped in three main families differing by the number of H-bonds. These measurements were repeated at different concentrations of trifluoroethanol (TFE)-water mixture, an alpha-helical promoting solvent, in order to check the stability of the conformations determined; no changes were observed up to 50% TFE in solution. Also temperature changes did not produce any variation in the ROESY spectrum in the same condition as above. The NMR and circular dichroism data strongly indicate the presence of an ordered (not alpha-helix nor beta-sheet) highly flexible structure in the eight amino acids repetitive units of SPR3, confirming the prediction of one possible beta-turn per each repeating unit. Thus, biochemical and biophysical data, strongly support SPR3 to function as a flexible cross-bridging protein to provide tensile strength or rigidity to the CE of the stratified squamous epithelia in which it is expressed.

Transglutaminase cross-linking properties of the small proline-rich 1 family of cornified cell envelope proteins. Integration with loricrin.

Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no alpha or beta structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.

Structural and transglutaminase substrate properties of the small proline-rich 2 family of cornified cell envelope proteins.

The small proline-rich (SPR) proteins are components of the cornified cell envelope of stratified squamous epithelia and become cross-linked to other proteins by transglutaminases (TGases). The SPR2 family is the most complex, as it consists of several differentially expressed members of the same size. To explore their physical and cross-linking properties, we have expressed in bacteria a human SPR2 family member, and purified it to homogeneity. By circular dichroism, it possesses no alpha or beta structure but has some organized structure associated with the central peptide repeat domain. The TGase 1, 2, and 3 enzymes expressed in epithelia use the recombinant SPR2 protein as a complete substrate in vitro, but with widely differing kinetic efficiencies, and in different ways. With TGase 1, only one glutamine on the head domain and one lysine on the tail domain were used for limited interchain cross-linking. With TGase 3, multiple head and tail domain residues were used for extensive interchain cross-linking. The total usage of glutamine and lysine residues in vitro by TGase 3 was similar to that seen in earlier in vivo studies. We conclude that SPR2 proteins are cross-linked in epithelia primarily by the TGase 3 enzyme, a minor extent by TGase 1, and probably not by TGase 2.

A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases.

We have addressed the question of how keratin intermediate filaments are associated with the cell envelope at the periphery of cornified epidermal cells. Many peptides from human epidermal cell envelopes containing isopeptide crosslinks inserted by transglutaminases in vivo have been characterized. A major subset involves the type II keratin chains keratin 1, 2e, 5, or 6 crosslinked to several protein partners through a lysine residue located in a conserved region of the V1 subdomain of their head domains. This sequence specificity was confirmed in in vitro crosslinking experiments. Previously the causative mutation in a family with diffuse nonepidermolytic palmar-plantar keratoderma was shown to be the loss in one allele of the same lysine residue of the keratin 1 chain. Ultrastructural studies of affected palm epidermis have revealed abnormalities in the organization of keratin filaments subjacent to the cell envelope and in the shape of the cornified cells. Together, these data suggest a mechanism for the coordination of cornified cell structure by permanent covalent attachment of the keratin intermediate filament cytoskeleton to the cell envelope by transglutaminase crosslinking. Furthermore, these studies identify the essential role of a conserved lysine residue on the head domains of type II keratins in the supramolecular organization of keratin filaments in cells.

The fate of trichohyalin. Sequential post-translational modifications by peptidyl-arginine deiminase and transglutaminases.

Trichohyalin (THH) is a major structural protein of the inner root sheath cells and medulla layer of the hair follicle and, to a lesser extent, of other specialized epithelia. THH is a high molecular weight insoluble alpha-helix-rich protein that forms rigid structures as a result of postsynthetic modifications by two Ca2+-dependent enzymes, transglutaminases (TGases) (protein cross-linking) and peptidyl-arginine deiminase (conversion of arginines to citrullines with loss of organized structure). The modified THH is thought to serve as a keratin intermediate filament matrix protein and/or as a constituent of the cell envelope. In this paper, we have explored in vitro the order of processing of THH to fulfill these functions, using an expressed truncated, more soluble form THH-8. THH-8 is a complete substrate for three known TGases expressed in epithelia, but the kinetic efficiency with TGase 3 is by far the greatest. Following maximal conversion of its arginines to citrullines, THH-8 is cross-linked even more efficiently by TGase 3, using most glutamines partially and all lysines. In addition, we show that insoluble aggregates of THH-8 or native pig tongue THH can be solubilized following peptidyl-arginine deiminase modification. Together, these data suggest an in vivo model in which THH located in insoluble cytoplasmic droplets is first modified by peptidyl-arginine deiminase which denatures it and makes it more soluble. This renders it available for efficient cross-linking by TGase 3 to form highly cross-linked rigid structures in the cells. This temporal order of reaction is supported by the observation that THH is expressed in hair follicle cells before the TGase 3 enzyme.

Sjögren-Larsson syndrome is caused by a common mutation in northern European and Swedish patients.

Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by congenital ichthyosis, mental retardation, and spastic diplegia or tetraplegia. Patients with SLS have deficient activity of fatty aldehyde dehydrogenase (FALDH), an enzyme involved in long-chain fatty alcohol oxidation. The cDNA encoding FALDH has recently been cloned and several different mutations have been found in SLS patients. We have now identified a point mutation (C943 --> T) in 7 of 19 kindreds of European descent, accounting for 24% of the SLS alleles. The C943T mutation was only found in patients of northern European ancestry from Sweden, the Netherlands, Germany, and Belgium. Haplotype analysis suggested that the patients carrying the C943T allele were distantly related. All four Swedish patients were homozygous for C943T, indicating that this mutation is probably the major cause of SLS in the inbred Swedish families. The mutation leads to the substitution of serine for the highly conserved proline 315 in the FALDH protein, and expression studies confirm that it destroys enzymatic activity. The mutation was readily detected with an MnlI restriction enzyme digestion test. The finding that C943T is a common SLS mutation in northern European and Swedish patients affords a rapid simple method for diagnosing SLS by screening patients for this mutation with DNA-based methods.

Protein unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships of the natural substrates trichohyalin and filaggrin.

Peptidylarginine deiminases, which are commonly found in mammalian cells, catalyze the deimination of protein-bound arginine residues to citrullines. However, very little is known about their substrate requirements and the significance or consequences of this postsynthetic modification. We have explored this reaction in vitro with two known substrates filaggrin and trichohyalin. First, the degree and rate of modification of arginines to citrullines directly correlates with the structural order of the substrate. In filaggrin, which has little structural order, the reaction proceeded rapidly to >95% completion. However, in the highly alpha-helical protein trichohyalin, the reaction proceeded slowly to about 25% and could be forced to a maximum of about 65%. Second, the rate and degree of modification depends on the sequence location of the target arginines. Third, we show by gel electrophoresis, circular dichroism, and fluorescence spectroscopy that the reaction interferes with organized protein structure: the net formation of >/=10% citrulline results in protein denaturation. Cyanate modification of the lysines in model alpha-helix-rich proteins to homocitrullines also results in loss of organized structure. These data suggest that the ureido group on the citrulline formed by the peptidylarginine deiminase enzyme modification functions to unfold proteins due to decrease in net charge, loss of potential ionic bonds, and interference with H bonds.

Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein.

Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier. In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a full-length cDNA clone in bacteria and purified it to homogeneity. We have also isolated loricrin from newborn mouse epidermis. By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no alpha or beta structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature. The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different. The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain cross-links involving different lysines and glutamines. Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo. The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo. Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene.

High amount of epsilon-(gamma-glutamyl)lysine cross-links in Mallory bodies.

Alcoholic hepatitis, the most severe form of alcoholic liver disease, is associated with inflammation, liver cell necrosis, and the appearance of Mallory bodies (MBs) in hepatocytes. Identical MBs can be experimentally induced in mouse livers by chronic griseofulvin or 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment. MBs are filamentous cytoplasmic inclusions containing insoluble high molecular weight protein material. Covalent polymerization of intracellular proteins may occur through formation of epsilon-(gamma-glutamyl)lysine cross-links catalyzed by Ca(2+)-dependent transglutaminases. Therefore, isolated experimentally-induced MBs were analyzed for the presence of epsilon-(gamma-glutamyl)lysine bonds. Highly purified MBs contained 19.7 nmole (griseofulvin-induced) and 14.4 nmoles (3,5-diethoxycarbonyl-1,4-dihydrocollidine induced) of isodipeptide linkage, respectively, per mg of protein. These results suggest that transglutaminase-induced cross-linking of proteins plays a major role in MB formation.

Presence and possible origin of epsilon(gamma-glutamyl)lysine isodipeptide in human plasma.

epsilon(gamma-glutamyl)lysine isodipeptide has been detected in normal human plasma by a sensitive HPLC technique in a concentration of 1.9-3.6 mumol/l. Incubation of in vitro clotted plasma at 37 degrees C for 12 h resulted in an increased amount of isodipeptide, and there was no further significant change when streptokinase was also present. Increased in vivo isodipeptide concentrations were also observed in hypercoagulable states and during fibrinolytic therapy.

Degradation of cells dying by apoptosis leads to accumulation of epsilon(gamma-glutamyl)lysine isodipeptide in culture fluid and blood.

epsilon(gamma-Glutamyl)lysine isodipeptide, the end-product of proteolytic digestion of proteins cross-linked by transglutaminase, was detected in culture fluid of neonatal rat hepatocytes and plasma of adult rats. The concentration of the isodipeptide was significantly increased in both when high rate of apoptosis with phagocytosis of dying hepatocytes was produced either by epidermal growth factor in the culture or by lead nitrate-induced hyperplasia with subsequent involution in rats. Specific induction of tissue transglutaminase and the consequent formation of highly cross-linked protein envelopes in apoptotic cells have been previously demonstrated by us in both systems.

The presence of a covalently cross-linked matrix in human platelets.

Exhaustive extraction of human platelets with 6 M guanidine-HCl, and 5% beta-mercaptoethanol, followed by 5% SDS resulted in a sedimentable material which showed fibrous structure by transmission electron microscopy. When platelets treated with 8 M urea, 50 mM DTT and 2% SDS were applied on a 3% solubilizable acrylamide gel a high molecular weight material could be also isolated which was highly crosslinked by epsilon(gamma-glutamyl)lysine bonds. Its amino acid composition was Asp 110, Glu 119, Ser 55, Gly 70, Arg 33, Thr 41, Ala 112, Pro 93, Tyr 35, Val 18, Met 55, Cys 46, IIe 47, Leu 71, Phe 27, Lys 76 expressed as residue per 1000. The quantity of platelet polymer material as well as the amount of epsilon(gamma-glutamyl)lysine bond was slightly higher in thrombin activated platelets. The insoluble matrix of resting platelets reacts with antibodies against spectrin, alpha-actinin, actin, myosin, tropomyosin. The matrix from activated platelets has shown reaction with additional antibodies including ones against blood coagulation factor XIIIa, fibrinogen, von Willebrand factor, thrombospondin, tubulin and filamin. The presence of an epsilon(gamma-glutamyl)lysine cross-linked cell matrix in platelets is consistent with the observation of a similar structure in other cells.

Protein cross-linking by transglutaminase induced in long-term potentiation in the Ca1 region of hippocampal slices.

Long-term potentiation induced by high-frequency stimulation of Schaffer collaterals in slices of rat hippocampus is accompanied by protein cross-linking by the Ca(2+)-dependent enzyme transglutaminase. This conclusion was drawn from the accumulation of the "isodipeptide" epsilon(gamma-glutamyl)lysine in the proteolytic digests of tetanized, but not of control, slices. The isopeptide bond is formed by transglutaminase between glutamyl-gamma-CONH2 and lysyl-epsilon-NH2 groups of proteins. It is suggested that the Ca(2+-induced covalent cross-linking of neuronal, probably dendritic, proteins may be part of the mechanism of long-term plastic changes via stabilization of newly formed supramolecular protein assemblies at the synapse.

Determination of epsilon (gamma-glutamyl)lysine crosslink in proteins using phenylisothiocyanate derivatization and high-pressure liquid chromatographic separation.

A sensitive, reproducible high-performance liquid chromatographic method is reported for the determination of the epsilon (gamma-glutamyl)lysine isopeptide bond, which is usually formed by protein-crosslinking transglutaminases between polypeptide chains. The procedure is based on the separation and quantitation of epsilon (gamma-glutamyl)lysine isodipeptide following exhaustive proteolytic digestion of the crosslinked peptide. It involves preliminary separation steps on a cation exchanger resin and a silica HPLC column, precolumn derivatization with phenylisothiocyanate, and reversed-phase high-pressure liquid chromatographic separation on a C18 column. The derivatized isodipeptide gave a linear concentration-response relationship, with a detection limit of 10 pmol/mg of protein. The combination of the preliminary separation steps and the sensitive detection system permits the determination of the epsilon (gamma-glutamyl)lysine crosslink in complex biological systems including total tissue homogenates.

Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.

N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.

Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action.

Physiological deletion of cells ensues programmed death which involves formation of apoptotic bodies with fragmented DNA. Here we report that apoptotic hepatocytes are insoluble in detergents, urea, guanidine hydrochloride, reducing agents and thereby can be isolated from rat liver following collagenase treatment. They are wrinkled, spherical structures similar to cornified envelopes of epidermis by phase-contrast microscopy and show irregular, globular morphology by scanning-electron microscopy. Part of their DNA content is cleaved into nucleosomal and oligonucleosomal fragments. Their insolubility, like that of the cornified envelope, is evoked by epsilon-(gamma-glutamyl)lysine and N1,N8-bis(gamma-glutamyl)spermidine protein cross-linking bonds formed by transglutaminase.