Josef Kellermann

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.

Lateral Gene Transfer of Dissimilatory (Bi)Sulfite Reductase Revisited

In contrast to previous findings, we demonstrate that the dissimilatory (bi)sulfite reductase genes (dsrAB) of Desulfobacula toluolica were vertically inherited. Furthermore, Desulfobacterium anilini and strain mXyS1 were identified, by dsrAB sequencing of 17 reference strains, as members of the donor lineage for those gram-positive Desulfotomaculum species which laterally acquired dsrAB.

Marine sponge collagen: isolation, characterization and effects on the skin parameters surface-pH, moisture and sebum

A previously described isolation procedure for collagen of the marine sponge Chondrosia reniformis Nardo was modified for scaling-up reasons yielding 30% of collagen (freeze-dried collagen in relation to freeze-dried sponge). Light microscope observations showed fibrous structures. Transmission electron microscopy studies proved the collagenous nature of this material: high magnifications showed the typical periodic banding-pattern of collagen fibres. However, the results of the amino acid analysis differed from most publications, presumably due to impurities that still were present. In agreement with earlier studies, sponge collagen was insoluble in dilute acid mediums and all solvents investigated. Dispersion of collagen was facilitated when dilute basic mediums were employed. The acid-base properties of the material were investigated by titration. Furthermore, a sponge extract was incorporated in two different formulations and compared with their extract-free analogues and a commercially available collagen containing product with respect to their effects on biophysical skin parameters. None of the preparations had a noticeable influence on the physiological skin surface pH. Skin hydration increased only slightly. However, all tested formulations showed a significant increase of lipids measured by sebumetry.

Human plasma kininogens are identical with α-cysteine proteinase inhibitors: Evidence from immunological, enzymological and sequence data

Human high‐ and low‐M r kininogens were shown to be potent inhibitors of cysteine proteinases such as cathepsin L and papain (K i = 17–48 pM). A strong immunological cross‐reaction between the kininogens and low‐M r α‐cysteine proteinase inhibitor from human plasma was found. Comparison of partial amino acid sequences from high‐ and low‐M r kininogen and low‐M r α‐cysteine proteinase inhibitor demonstrated sequence identity for all segments analyzed. These findings suggest that the kininogens and the α‐cysteine proteinase inhibitors from human plasma are identical proteins.Human high- and low-Mr kininogens were shown to be potent inhibitors of cysteine proteinases such as cathepsin L and papain (Ki = 17-48 pM). A strong immunological cross-reaction between the kininogens and low-Mr alpha-cysteine proteinase inhibitor from human plasma was found. Comparison of partial amino acid sequences from high- and low-Mr kininogen and low-Mr alpha-cysteine proteinase inhibitor demonstrated sequence identity for all segments analyzed. These findings suggest that the kininogens and the alpha-cysteine proteinase inhibitors from human plasma are identical proteins.

HDF2, the Second Subunit of the Ku Homologue from Saccharomyces cerevisiae

The high affinity DNA binding factor (HDF) protein of Saccharomyces cerevisiae is composed of two subunits and specifically binds ends of double-stranded DNA. The 70-kDa subunit, HDF1, shows significant homology with the 70-kDa subunit of the human Ku protein. Like the Ku protein, HDF1 has been shown to be involved in recombination and double stranded DNA break repair. We have purified and cloned HDF2, the second subunit of the HDF protein. The amino acid sequence of HDF2 shows a 45.6% homology with the 80-kDa subunit of the Ku protein. HDF1 by itself does not bind DNA, while HDF2 protein on its own seems to displays DNA binding activity. Targeted disruption of the HDF2 gene causes a temperature-sensitive phenotype for growth comparable to the phenotype of hdf1− strains. The human Ku protein cannot complement this temperature-sensitive phenotype. hdf2− strains are sensitive to bleomycin and methyl methanesulfonate, but this sensitivity is reduced in comparison with hdf1− strains.

The thermostable alpha-L-rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial alpha-L-rhamnoside hydrolase, a new type of inverting glycoside hydrolase.

An α‐l‐rhamnosidase clone was isolated from a genomic library of the thermophilic anaerobic bacterium Clostridium stercorarium and its primary structure was determined. The recombinant gene product, RamA, was expressed in Escherichia coli, purified to homogeneity and characterized. It is a dimer of two identical subunits with a monomeric molecular mass of 95 kDa in SDS polyacrylamide gel electrophoresis. At pH 7.5 it is optimally active at 60°C and insensitive to moderate concentrations of Triton X100, ethanol and EDTA. It hydrolysed p‐nitrophenyl‐α‐l‐rhamnopyranoside, naringin and hesperidin with a specific activity of 82, 1.5 and 0.46 U mg−1 respectively. Hydrolysis occurs by inversion of the anomeric configuration as detected using 1H‐NMR, indicating a single displacement mechanism. Naringin was hydrolysed to rhamnose and prunin, which could further be degraded by incubation with a thermostable β‐glucosidase. The secondary structure of RamA consists of 27% α‐helices and 50% β‐sheets, as detected by circular dichroism. The primary structure of the ramA gene has no similarity to other glycoside hydrolase sequences and possibly is the first member of a new enzyme family.

Genealogy of mammalian cysteine proteinase inhibitors: Common evolutionary origin of stefins, cystatins and kininogens

A model for the evolution of mammalian cysteine proteinase inhibitors has been constructed on the basis of sequence homology. This model suggests that the diversity of cysteine proteinase inhibitors has evolved from two ancestral units forming the building blocks of stefin and cystatin. Gene triplication of the archetypal inhibitor generated the kininogen heavy chain which contains three cystatin-like copies. Hence, the superfamily of mammalian cysteine proteinase inhibitors is constituted by at least three distinct families, with stefin, cystatin and kininogen as their prototypes.A model for the evolution of mammalian cysteine proteinase inhibitors has been constructed on the basis of sequence homology. This model suggests that the diversity of cysteine proteinase inhibitors has evolved from two ancestral units forming the building blocks of stefin and cystatin. Gene triplication of the archetypal inhibitor generated the kininogen heavy chain which contains three cystatin‐like copies. Hence, the superfamily of mammalian cysteine proteinase inhibitors is constituted by at least three distinct families, with stefin, cystatin and kininogen as their prototypes.

Inhibition of the Interaction of Urokinase-Type Plasminogen Activator (uPA) with Its Receptor (uPAR) by Synthetic Peptides

Focusing of the serine protease urokinase-type plasminogen activator (uPA) to the cell surface via interaction with its specific receptor (uPAR, CD87) is an important step for tumor cell invasion and metastasis. The ability of a synthetic peptide derived from the uPAR-binding region of uPA (comprising amino acids 16-32 of uPA; uPA(16-32)) to inhibit binding of fluorescently labeled uPA to uPAR on human promyeloid U937 cells was assessed by quantitative flow cytofluorometric analysis (FACS) and compared to the inhibitory capacities of other synthetic peptides known to interfere with uPA/uPAR-interaction. An about 3000-fold molar excess of uPA(16-32) resulted in 50% inhibition of pro-uPA binding to cell surface-associated uPAR. Using a solid-phase uPA-ligand binding assay employing recombinant soluble uPAR coated to microtiter plates, the minimal binding region of wild-type uPA was determined. The linear peptide uPA(19-31) and its more stable disulfide-bridged cyclic form (cyclo(19,31)uPA(19-31)) displayed uPAR-binding activity whereas other peptides such as uPA(18-30), uPA(20-32) or uPA(20-30) did not react with uPAR. Cyclic peptide derivatives of cyclo(19,31)uPA(19-31) in which certain amino acids were deleted and/or replaced by other amino acids as well as uPAR-derived wild-type peptides did also not inhibit uPA/uPAR-interaction. Therefore, the present investigations identified cyclo(19,31)uPA(19-31) as a potential lead structure for the development of uPA-peptide analogues to block uPA/uPAR-interaction.

High molecular weight aspartic endopeptidase generates a coronaro-constrictory peptide from the β-chain of hemoglobin

Studying the influence of brain cathepsin D (EC 3.4.23.5) and high molecular weight (HMW) aspartic endopeptidase (EC 3.4.23.‐) on the processing of hypothalamic calmodulin‐binding coronaro‐constrictory peptide factors from the β‐chain of globin it was found that only HMW aspartic endopeptidase generates the fragment 31–40 of the β‐chain of bovine hemoglobin (Hb) by cleavage of the Leu30‐Leu31 and Phe40‐Phe41 bonds. Digestion of the β‐chain of globin was performed at 37°C at an enzyme/substrate ratio of 1:80 at pH 3.5 using different times of incubation (from 4 h to 10 h). The resulting peptides were separated by reversed‐phase high‐performance liquid chromatography (HPLC) and then identified by amino acid analysis and Edman degradation. The differences in specificity and activity of these two brain aspartic proteinases could be explained by their different structural features. Our finding provides evidence for a different biological function of these two enzymes. Data obtained give us reason to suppose that HMW aspartic proteinase probably can participate in the processing of the coronaro‐constrictory peptide in vivo by limited proteolysis of Hb or Hb‐like protein.

Interplay of human tissue kallikrein 4 (hK4) with the plasminogen activation system: hK4 regulates the structure and functions of the urokinase-type plasminogen activator receptor (uPAR)

Abstract The plasminogen activation system is involved in cancer progression and metastasis. Among other proteolytic factors, it includes the serine protease urokinase-type plasminogen activator (uPA) and its three-domain (D1D2D3) receptor uPAR (CD87), which focuses plasminogen activation to the cell surface. The function of uPAR is regulated in part through shedding of domain D1 by proteases, e.g., uPA itself or plasmin. Human tissue kallikrein 4 (hK4), which is highly expressed in prostate and ovarian tumor tissue, was previously shown to cleave and activate the pro-enzyme forms of prostate-specific antigen (PSA, tissue kallikrein hK3) and uPA. Here we demonstrate that uPAR is also a target for hK4, being cleaved in the D1-D2 linker sequence and, to a lesser extent, in its D3 juxtamembrane domain. hK4 may thus modulate the tumor-associated uPA/uPAR-system activity by either activating the pro-enzyme form of uPA or cleaving the cell surface-associated uPA receptor.