J. Vandekerckhove

Protein-chemical characterization of NF-H, the largest mammalian neurofilament component; intermediate filament-type sequences followed by a unique carboxy-terminal extension

NF‐H has the highest mol. wt. of the three mammalian neurofilament components (NF‐L, NF‐M, NF‐H). In spite of its unusually large mol. wt., estimated to be 200 K by gel electrophoresis, NF‐H contains sequences which identify it as an integral intermediate filament (IF) protein in its amino‐terminal region. We have isolated and partially characterized a basic, non‐α‐helical segment located at the amino‐terminal end with properties similar to headpieces of other non‐epithelial IF proteins. The highly α‐helical 40‐K fragment excised by chymotrypsin is now identified by the amino acid sequence of a 17‐K fragment. This sequence can be unambiguously aligned with the rod region of other IF proteins and covers about half of the presumptive coiled‐coil arrays. NF‐H and NF‐M show 45% sequence identity in this region. The extra mass of NF‐H in comparison with most other IF proteins arises from a carboxy‐terminal extension thought to be responsible for inter‐neurofilament cross‐bridges in axons. This autonomous domain has a unique amino acid composition characterized by a high content of proline, alanine and particularly of lysine and glutamic acid. The NF‐H tailpiece extension also carries a large number of serine phosphates, which are not evenly distributed, but are restricted to the amino‐terminal part. Having now delineated the intermediate filament‐type sequences for all three neurofilament proteins it seems very likely that the three components interact via coiled‐coil interactions. They all carry unique carboxy‐terminal extensions which increase in length from NF‐L to NF‐H and seem to extend from the filament wall.

Complete nucleotide sequence coding for rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase derived from a cDNA clone

cDNA clones for 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase were isolated from rat liver expression libraries in λgt11 by antibody, oligonucleotide, and cDNA screening. One 1860 bp long clone contained a full‐length nucleotide sequence coding for the 470 amino acids of each of the two identical subunits of the bifunctional enzyme. This clone also contained untranslated sequences, one 173 bp long upstream from the ATG start codon and one 271 bp long downstream from the TGA stop codon. The clone was terminated by a poly(A) tail of 29 nucleotides.

Protein-Electroblotting and Microsequencing in Establishing Integrated Human Protein Databases

Proteins, which are characteristic for a specific state of differentiation, the transformed phenotype or pathological conditions of human cells and tissues were identified by computer analyzed two-dimensional gel electrophoresis. Sequenceable amounts of protein were collected from multiple gels with a gel-concentration device, enabling the elution and concentration of more than twenty protein spots, suspended in 1 ml of sample buffer. The eluted protein was concentrated in a new gel in a very small spot and then electroblotted onto polybase-coated glass-fiber or polyvinylidene-difluoride membranes and in situ digested. The released peptides were separated by micro-bore or narrow-bore reversed phase HPLC and immediately collected on polyethylenimine-coated glass-fiber discs for sequencing. These variations of previously developed methods allowed us to work at higher sensitivity. The procedure is currently being used to try out a systematic analysis of human proteins recovered from two-dimensional gels.

Porcine vinculin and metavinculin differ by a 68-residue insert located close to the carboxy-terminal part of the molecule.

Metavinculin is a higher mol. wt variant of vinculin expressed only in muscle tissue. Using amino acid sequencing methods on the intact molecules and their proteolytic subfragments, together with a polyclonal antibody specific only for metavinculin from porcine stomach, we have been able to identify and sequence the difference peptide in the porcine metavinculin molecule. By alignment with the complete sequence of chick fibroblast vinculin (communicated by G.J. Price, P. Jones, M.D. Davison, R. Bendori, S. Griffiths, B. Patel, B. Geiger and D.R. Critchley, prior to publication) the exact location of the insert could be determined. In porcine metavinculin, this insert lies between the 90‐kd protease‐resistant amino‐terminal core and the carboxy terminus of the molecule. It contains 68 amino acids and is flanked by KWSSK sequences, one of which is present in vinculin. The identity of the mapped vinculin and metavinculin sequences outside this difference peptide is consistent with the two proteins arising via alternative splicing at the mRNA level. The lack of reactivity of the porcine metavinculin antibody with metavinculin from chicken as well as the finding of different proteolytic cleavage sites in avian metavinculin indicate a species‐specific amino acid sequence in the difference piece of the metavinculin molecule.

Studies of the Role of the Propeptides of the Arabidopsis thaliana 2s Albumin

To investigate the possible roles of the Arabidopsis thaliana 2S albumin propeptides with respect to sorting, processing, and stability of the protein in plant cells, five gene constructions deleting or modifying the propeptides were made based on one of the genes encoding the Arabidopsis 2S albumin. These constructions were introduced into tobacco (Nicotiana tabacum) plants. Using subcellular fractionation and immunocytochemistry on ripe seeds, it was demonstrated that none of the propeptides was necessary for the sorting of the protein. Detailed protein-chemical analysis of the mature gene products indicated that, for all of the modified 2S albumin precursors made, the proteins were stably folded and correctly processed. However, the latter is less efficient when the internal fragment between the small and the large subunit is missing or when this internal fragment is changed. In an attempt to establish a rapid assay system for modified 2S albumin precursors, yeast cells were transformed with the same gene constructs. It was demonstrated that the processing machinery in yeast cells differs from that in plants, and, in a perhaps related observation, differences in stability of a particular modified protein were observed.

Tumour necrosis factor induces phosphorylation primarily of the nitric-oxide-responsive form of glyoxalase I

We have previously shown that TNF (tumour necrosis factor) induces phosphorylation of GLO1 (glyoxalase I), which is required for cell death in L929 cells. In the present paper, we show that the TNF-induced phosphorylation of GLO1 occurs primarily on the NO (nitric oxide)-responsive form of GLO1. In addition, analysis of several cysteine mutants of GLO1 indicated that Cys-138, in combination with either Cys-18 or Cys-19, is a crucial target residue for the NO-mediated modification of GLO1. Furthermore, the NO-donor GSNO (S-nitrosogluthathione) induces NO-mediated modification of GLO1 and enhances the TNF-induced phosphorylation of this NO-responsive form. GSNO also strongly promotes TNF-induced cell death. By the use of pharmacological inhibition of iNOS (inducible NO synthase) and overexpression of mutants of GLO1 that are deficient for the NO-mediated modification, we have shown that the NO-mediated modification of GLO1 is not a requirement for TNF-induced phosphorylation or TNF-induced cell death respectively. In summary, these data suggest that the TNF-induced phosphorylation of GLO1 is the dominant factor for cell death.

The role of adhesive F107 fimbriae and of SLT-IIv toxin in the pathogenesis of edema disease in pigs

Colonization of the small intestine and the excretion of a toxin are important steps in the pathogenesis of edema disease in pigs. Although much is known about the chemical and biological characteristics of SLT-IIv toxin, its mode of action and its genetic determinant, F107 fimbriae were only recently described as colonization factors. Here we summarize our current knowledge about the virulence factors F107 fimbriae and SLT-IIv toxin.

The role of vacuolar and secreted pathogenesis-related ß (1–3)-glucanases and chitinases in the defence response of plants

SummaryUpon infection of a plant by a pathogen, a series of drastic metabolic changes occur within the plant. One characteristic feature of this defence response is the synthesis of the so-called pathogenesis-related (PR) proteins. We have studied the nature, structure, and subcellular localization of the different PR proteins upon salicylic acid treatment and Pseudomonas syringae infection of Nicotiana tabacum plants. In both test systems, we could demonstrate that the PR protein fraction of tobacco consists of at least 20 to 25 different proteins, including s (1–3)-glucanases, chitinases, peroxidases, thaumatin-like proteins, the PR1 class proteins and a proteinase inhibitor-like protein. Moreover, several classes of these PR proteins segregate into specific vacuolar and secreted isoforms. Here, we present a model which could explain the role of the compartimentalized PR s (1–3)-glucanases and chitinases within the regulation of the defence response.

Microsequencing of plant proteins and cloning of the corresponding genes by oligonucleotide probing

The production of secondary metabolites in plants requires the coordinate and developmentally regulated expression of a set of genes. The understanding of the molecular mechanisms underlying the spatial expression of plant genes such as these involved in secondary product biosynthesis is emerging as one of the most exciting fields in plant molecular biology research. Essential for such an analysis is the ability to clone the appropriate genes. In this chapter, we report the development of a powerful new approach for isolating plant genes whose the expression is correlated with certain phenotypes or environmental conditions. This approach is based on the ability of obtaining N-terminal amino-acid sequences from proteins which are immobilized on glass-fiber sheets coated with a polybase. Subsequently, oligonucleotides matching the amino-acid sequences are used to clone corresponding cDNA clones.

Molecular domain structure of porcine vinculin and metavinculin

SummaryMetavinculin is a higher molecular weight variant of vinculin expressed only in cardiac and smooth muscle. Using microsequencing methods on the intact molecules and their proteolytic subfragments we have been able to map the common and different parts of these closely related proteins. Both vinculin and metavinculin, from mammals and birds exhibit a relatively protease resistant 90 kD core fragment. N-terminal sequencing analysis of the avian and mammalian core fragments as well as of major core subfragments obtained by extended proteolysis placed the core domain at the N-terminus of the intact molecules and revealed identity between metavinculin and vinculin as well as between species. Limited chymotryptic digestion of porcine vinculin and metavinculin yielded a common 16 kD fragment which could be placed at the C-terminus of the cDNA sequence derived from chick fibroblast vinculin (G. J.Price, P.Jones, M. D.Davison, R.Bendori, S.Griffiths, B.Patel, B.Geiger and D. R.Critchley 1988, in press). From additional sequence data the metavinculin specific fragment could be placed at the metavinculin C-terminus. Using a polyclonal antibody specific for porcine metavinculin a peptide unique to metavinculin could be identified. Direct sequencing of this, as well as of related, overlapping fragments, purified by reversed phase HPLC revealed a 68 amino acid insert in procine metavinculin, between the core fragment and the C-terminal piece, common to vinculin and metavinculin. The domain organizazions of vinculin and metavinculin and their possible functional implications are discussed.