Reinhard Mentele

Matching of oligoclonal immunoglobulin transcriptomes and proteomes of cerebrospinal fluid in multiple sclerosis

We describe a method for correlating the immunoglobulin (Ig) proteomes with the B cell transcriptomes in human fluid and tissue samples, using multiple sclerosis as a paradigm. Oligoclonal Ig bands and elevated numbers of clonally expanded B cells in the cerebrospinal fluid (CSF) are diagnostic hallmarks of multiple sclerosis. Here we compared the Ig transcriptomes of B cells with the corresponding Ig proteomes in CSF samples from four subjects with multiple sclerosis. We created individual Ig transcriptome databases that contained the subject-specific mutations introduced by V(D)J recombination and somatic hypermutation and then searched the CSF for corresponding characteristic peptides by mass spectrometry. In each sample, the Ig transcriptomes and proteomes strongly overlapped, showing that CSF B cells indeed produce the oligoclonal Ig bands. This approach can be applied to other organ-specific diagnostic fluid or tissue samples to compare the Ig transcripts of local B cells with the corresponding antibody proteomes of individual subjects.

Myelin-specific T cells also recognize neuronal autoantigen in a transgenic mouse model of multiple sclerosis

We describe here the paradoxical development of spontaneous experimental autoimmune encephalomyelitis (EAE) in transgenic mice expressing a myelin oligodendrocyte glycoprotein (MOG)-specific T cell antigen receptor (TCR) in the absence of MOG. We report that in Mog-deficient mice (Mog−/−), the autoimmune response by transgenic T cells is redirected to a neuronal cytoskeletal self antigen, neurofilament-M (NF-M). Although components of radically different protein classes, the cross-reacting major histocompatibility complex I-Ab–restricted epitope sequences of MOG35–55 and NF-M18–30 share essential TCR contact positions. This pattern of cross-reaction is not specific to the transgenic TCR but is also commonly seen in MOG35–55–I-Ab–reactive T cells. We propose that in the C57BL/6 mouse, MOG and NF-M response components add up to overcome the general resistance of this strain to experimental induction of autoimmunity. Similar cumulative responses against more than one autoantigen may have a role in spontaneously developing human autoimmune diseases.

Related B cell clones that populate the CSF and CNS of patients with multiple sclerosis produce CSF immunoglobulin

We investigated the overlap shared between the immunoglobulin (Ig) proteome of the cerebrospinal fluid (CSF) and the B cell Ig-transcriptome of CSF and the central nervous system (CNS) tissue of three patients with multiple sclerosis. We determined the IgG-proteomes of CSF by mass spectrometry, and compared them to the IgG-transcriptomes from CSF and brain lesions, which were analyzed by cDNA cloning. Characteristic peptides that were identified in the CSF-proteome could also be detected in the transcriptomes of both, brain lesions and CSF, providing evidence for a strong overlap of the IgG repertoires in brain lesions and in the CSF.

Isolation, Cloning and Structural Characterisation of Boophilin, a Multifunctional Kunitz-Type Proteinase Inhibitor from the Cattle Tick

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca.

Mature P-III snake metalloproteinases are soluble venom components which belong to the Reprolysin sub family and are structurally related to the mammalian membrane-bound A Disintegrin And Metalloproteinase (ADAMs). Here we present the molecular cloning of bothropasin, a metalloproteinase with hemorrhagic and myonecrotic activities isolated from the venom of Bothrops jararaca. The full-length cDNA encoding the bothropasin precursor was cloned by immunoscreening and its authenticity was confirmed by the amino acid sequence of internal fragments obtained from an autolyzed sample of native bothropasin. The predicted bothropasin precursor is comprised of the elements of a P-III venom metalloproteinase: signal sequence, pro-, metalloproteinase, disintegrin-like and cysteine-rich domains. In the autolysis process of native bothropasin, the disintegrin-like and cysteine-rich domains remained intact while the metalloproteinase domain was cleaved at different sites. The attempts made to obtain the recombinant precursor form of bothropasin using bacterial, yeast and mammalian cell expression systems failed to produce it in an amount sufficient to analyze the activation of the zymogen. Nevertheless, the study of the expression of the individual domains of bothropasin using a bacterial system resulted in the production of recombinant pro-and disintegrin-like+cysteine-rich domains but not the metalloproteinase domain. These results along with the autolysis pattern of the native protein suggest a role for the metalloproteinase domain in the structural stability of bothropasin.

Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins

Significance Oligoclonal bands (OCBs) of the cerebrospinal fluid (CSF) are a hallmark of multiple sclerosis (MS). They are expanded antibody species that are detectable in >95% of patients. Because several OCB and polyclonal antibodies are present in a CSF sample, it was for technical reasons thus far not possible to isolate distinct OCBs and identify their antigens. Here we combined refined biochemical analysis, proteomics, and transcriptomics to molecularly characterize distinct OCB antibodies. We produced six recombinant OCB antibodies and characterized three autoantigens. All of them were ubiquitous intracellular proteins, not specific to brain tissue. This finding indicates that in MS, part of the OCBs do not directly mediate tissue destruction, but rather, indicate a secondary immune response. Oligoclonal Ig bands (OCBs) of the cerebrospinal fluid are a hallmark of multiple sclerosis (MS), a disabling inflammatory disease of the central nervous system (CNS). OCBs are locally produced by clonally expanded antigen-experienced B cells and therefore are believed to hold an important clue to the pathogenesis. However, their target antigens have remained unknown, mainly because it was thus far not possible to isolate distinct OCBs against a background of polyclonal antibodies. To overcome this obstacle, we copurified disulfide-linked Ig heavy and light chains from distinct OCBs for concurrent analysis by mass spectrometry and aligned patient-specific peptides to corresponding transcriptome databases. This method revealed the full-length sequences of matching chains from distinct OCBs, allowing for antigen searches using recombinant OCB antibodies. As validation, we demonstrate that an OCB antibody from a patient with an infectious CNS disorder, neuroborreliosis, recognized a Borrelia protein. Next, we produced six recombinant antibodies from four MS patients and identified three different autoantigens. All of them are conformational epitopes of ubiquitous intracellular proteins not specific to brain tissue. Our findings indicate that the B-cell response in MS is heterogeneous and partly directed against intracellular autoantigens released during tissue destruction. In addition to helping elucidate the role of B cells in MS, our approach allows the identification of target antigens of OCB antibodies in other neuroinflammatory diseases and the production of therapeutic antibodies in infectious CNS diseases.

Amino acid sequence of a lectin-like protein from Lachesis muta stenophyrs venom

The primary structure of the lectin-like protein from Lachesis muta stenophyrs venom was deduced from analysis of the N-terminus and the sequence of peptides obtained after digestion with trypsin, Arg-C enzyme, Staphylococcus aureus V8 protease and endoproteinase Asp-N. Peptides generated by cleavage of the lectin with cyanogen bromide and o-iodosobenzoic acid were also sequenced. Comparison of the complete 135 amino acid residues sequence with those of the lectin from the venom of Crotalus atrox, with platelet coagglutinin from Bothrops jararaca beta-fragment and with the anticoagulant B protein chain from Trimeresurus flavoviridis venom, revealed 92, 46 and 29% identity, respectively. Significant homology was also found with C-type carbohydrate-recognition domain-like structures from invertebrate and vertebrate lectins. To our knowledge, this is the second known primary structure of a lectin-like protein from snake venom.

Structural characterization of coagulant Moringa oleifera Lectin and its effect on hemostatic parameters.

Lectins are carbohydrate recognition proteins. cMoL, a coagulant Moringa oleifera Lectin, was isolated from seeds of the plant. Structural studies revealed a heat-stable and pH resistant protein with 101 amino acids, 11.67 theoretical pI and 81% similarity with a M. oleifera flocculent protein. Secondary structure content was estimated as 46% α-helix, 12% β-sheets, 17% β-turns and 25% unordered structures belonging to the α/β tertiary structure class. cMoL significantly prolonged the time required for blood coagulation, activated partial thromboplastin (aPTT) and prothrombin times (PT), but was not so effective in prolonging aPTT in asialofetuin presence. cMoL acted as an anticoagulant protein on in vitro blood coagulation parameters and at least on aPTT, the lectin interacted through the carbohydrate recognition domain.

An Extracellular Tetrathionate Hydrolase from the Thermoacidophilic Archaeon Acidianus Ambivalens with an Activity Optimum at pH 1.

Background: The thermoacidophilic and chemolithotrophic archaeon Acidianus ambivalens is routinely grown with sulfur and CO2-enriched air. We had described a membrane-bound, tetrathionate (TT) forming thiosulfate:quinone oxidoreductase. Here we describe the first TT hydrolase (TTH) from Archaea. Results: A. ambivalens cells grown aerobically with TT as sole sulfur source showed doubling times of 9 h and final cell densities of up to 8 × 108/ml. TTH activity (≈0.28 U/mg protein) was found in cell-free extracts of TT-grown but not of sulfur-grown cells. Differential fractionation of freshly harvested cells involving a pH shock showed that about 92% of the TTH activity was located in the pseudo-periplasmic fraction associated with the surface layer, while 7.3% and 0.3% were present in the soluble and membrane fractions, respectively. The enzyme was enriched 54-fold from the cytoplasmic fraction and 2.1-fold from the pseudo-periplasmic fraction. The molecular mass of the single subunit was 54 kDa. The optimal activity was at or above 95°C at pH 1. Neither PQQ nor divalent cations had a significant effect on activity. The gene (tth1) was identified following N-terminal sequencing of the protein. Northern hybridization showed that tth1 was transcribed in TT-grown cells in contrast to a second paralogous tth2 gene. The deduced amino acid sequences showed similarity to the TTH from Acidithiobacillus and other proteins from the PQQ dehydrogenase superfamily. It displayed a β-propeller structure when being modeled, however, important residues from the PQQ-binding site were absent. Conclusion: The soluble, extracellular, and acidophilic TTH identified in TT-grown A. ambivalens cells is essential for TT metabolism during growth but not for the downstream processing of the TQO reaction products in S°-grown cells. The liberation of TTH by pH shock from otherwise intact cells strongly supports the pseudo-periplasm hypothesis of the S-layer of Archaea.

Human μ-calpain: Simple isolation from erythrocytes and characterization of autolysis fragments

Abstract Heterodimeric calpain, consisting of the large (80 kDa) and the small (30 kDa) subunit, was isolated and purified from human erythrocytes by a highly reproducible fourstep purification procedure. Obtained material is more than 95% pure and has a specific activity of 6 7 mU/mg. Presence of contaminating proteins could not be detected by HPLC and sequence analysis. During storage at 80 C the enzyme remains fully activatable by Ca 2+ , although the small subunit is partially processed to a 22 kDa fragment. This novel autolysis product of the small subunit starts with the sequence 60 RILG and is further processed to the known 18 kDa fragment. Active forms and typical transient and stable autolysis products of the large subunit were identified by protein sequencing. In caseinzymograms only the activatable forms 80 kDa+30 kDa, 80 kDa+22 kDa and 80 kDa+18 kDa displayed caseinolysis.