Knut Sletten

A novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas

Deposition of amyloid is the most constantly present alteration in the islets of Langerhans in type 2 diabetes mellitus and is also quite common in insulin-producing tumors of the pancreas and it is very likely that these two amyloids are identical. We have isolated amyloid fibrils from an insulin-secreting human tumour and purified the fibrillar protein. N-terminal amino acid sequence of the protein is unique and does not resemble insulin or its precursors. Instead it has about 50% homology with the neuropeptide CGRP (calcitonin gene related peptide).

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.

Evidence that the amyloid fibril protein in senile systemic amyloidosis is derived from normal prealbumin.

Familial amyloidosis in different kindreds is associated with a variety of point mutations in the prealbumin gene, resulting in prealbumin variants which are believed to be amyloidogenic, i.e. prone to form amyloid fibrils. In the most common amyloid-associated variant, there is a methionine for valine substitution in position 30. We have studied the prealbumin-derived amyloid protein ASc1 in the common age-related senile systemic amyloidosis. Evidence is presented that there is no abnormality in the primary structure of prealbumin in this disease and that, in addition to complete prealbumin, fibrils contain prealbumin fragments lacking a significant part of the N-terminus.

Amyloid deposits in transthyretin-derived amyloidosis: cleaved transthyretin is associated with distinct amyloid morphology

The pathological fibrillar deposits found in the heart and other organs of patients with senile systemic amyloidosis (SSA) and Swedish familial amyloidotic polyneuropathy (FAP) contain wild‐type (wt) and a mutant form of transthyretin (TTR), respectively. Previously, it was reported that these two forms of amyloid have different molecular features and it was thus postulated that the mechanism responsible for TTR fibrillogenesis in SSA and FAP may differ. To document further the nature of the amyloid in these entities, detailed morphological, histochemical, immunological, and structural analyses of specimens obtained from 14 individuals with SSA and 11 Swedish FAP patients have been performed. Two distinct patterns of amyloid deposition (designated A and B) were evident. In pattern A, found in all SSA and five of 11 FAP cases, the amyloid had a homogeneous but patchy distribution within the sub‐endocardium, sub‐epicardium, and myocardium; exhibited weak congophilia and green birefringence; and was composed of tightly packed, short, unorientated fibrils. This material contained mainly ∼79‐residue C‐terminal fragments of the amyloidogenic precursor protein. In pattern B, seen in the six other FAP patients, the amyloid appeared as thin streaks throughout the cardiac tissue; often surrounded individual muscle cells; was strongly congophilic and birefringent; had long fibrils arranged in parallel bundles, often penetrating into myocytes; and was composed of virtually intact TTR molecules. These findings provide substantive evidence for the morphological and structural heterogeneity of TTR fibrils and suggest that the two types of deposition may reflect fundamental differences in the pathogenesis of the TTR‐associated amyloidoses. Copyright

Oldenlandia affinis (R&S) DC. A plant containing uteroactive peptides used in African traditional medicine.

A review of the geographical distribution, clinical use, biological activity and phytochemistry of Oldenlandia affinis (R&S) DC. is presented. During an inventory of medicinal plants in northern Congo/Brazzaville and south-western Central African Republic in 1962, 196 different species were registered, one of which was O. affinis used for the facilitation of childbirth. A medical team working in Luluabourg (Kananga) in Congo during the troubled period in 1960, discovered also the traditional use of the same plant as an oxitocic agent during labour. The plant was collected and the uterotonic substances isolated. Cyclic peptides (called Kalata-peptides) were described, and the main peptide, B1, was subjected to pharmacological and chemical investigations. Later the three-dimensional structure of the peptide was determined. Similar cyclic peptides have been isolated also from other plants in the Rubiaceae family like Chassalia pasvifoloia and Psychotria longipes, and from Viola species: Viola tricolor L. and Viola arvensis Murray. Some of these peptides, included Kalata-peptide B1, have been shown to hold antimicrobial activity. They have recently been synthesized, and they may represent a starting point for the design of new peptide antibiotics.

Protein purification and gene isolation of chlamysin, a cold‐active lysozyme‐like enzyme with antibacterial activity

An antibacterial ∼11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram‐positive and Gram‐negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5–6.2) and at low temperature (4–35°C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70°C for 15 min, suggesting relatively high protein structure stability. Sequence‐analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme.

The Leucocyte L1 Protein: Identity with the Cystic Fibrosis Antigen and the Calcium-Binding MRP-8 and MRP-14 Macrophage Components

The partial amino acid sequence of L1 protein light and heavy chains reveals an overall structure identical to the two macrophage proteins, MRP‐8 and MRP‐14, deduced from the sequence of the cDNA encoding the polypeptides. The light chain of L1 protein (L1‐L) was shown to contain two modified amino acid residues.

Proteolysis of AA Amyloid Fibril Proteins by Matrix Metalloproteinases-1, -2, and -3

We recently demonstrated the presence of matrix metalloproteinases (MMPs)-1, -2, and -3 in AA amyloid deposits, which lead us to speculate that MMPs may participate in amyloidogenesis by either processing the precursor protein, or by degrading the amyloid deposits. Here we investigated this theory by determining the ability of MMP-1, -2, and -3 to degrade human acute-phase serum amyloid A (SAA) and human AA amyloid fibril proteins (AFPs). The following in vitro degradation experiments were performed: using either recombinant MMP-1, -2, or -3 and SAA as a substrate; using either recombinant MMP-1, -2, or -3 and AFP as a substrate; and using THP-1 cells as the protease source and AFP as the substrate. All three MMPs were able to cleave SAA and AFP within the region spanning residues 51 to 57. The following cleavage sites were identified: at 57 to 58 for MMP-1; at 7 to 8 and 51 to 52 for MMP-2; at 7 to 8, 16 to 17, 23 to 24, 51 to 52, 55 to 56, 56 to 57, and 57 to 58 for MMP-3. Cell culture experiments showed that THP-1 cells were able to degrade AFPs. Degradation was significantly delayed after addition of a general metalloproteinase inhibitor (o-phenanthroline) to dextran sulfate-stimulated cells. This is the first study to show that human SAAs and AFPs are susceptible to proteolytic cleavage by MMPs. Immunocytochemistry and electron microscopy showed that degradation takes place in the pericellular or extracellular compartment.

Purification and characterization of plantaricin A, a Lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides.

A Lactobacillus plantarum bacteriocin, plantaricin A, has been purified to homogeneity by ammonium sulphate precipitation, binding to cation exchanger and Octyl-Sepharose, and reverse-phase chromatography. The bacteriocin activity was associated with two peptides, termed alpha and beta, which were separated upon reverse-phase chromatography. Bacteriocin activity required the complementary action of both the alpha and beta peptides. From the N-terminal end, 21 and 22 amino acid residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acid residues, suggesting that either the C terminus had been reached or that modifications in the next amino acid residue blocked the sequencing reaction. Judging from their amino acid sequence, alpha and beta may be encoded by the same gene, since alpha appeared to be a truncated form of beta. Alanine, the first amino acid residue at the N-terminal end of beta was not present at this position in alpha. Otherwise the sequences of alpha and beta appeared to be identical. The calculated molecular masses of the sequenced part of alpha and beta were 2426 and 2497 Da, respectively. The molecular masses of alpha and beta as determined by mass spectroscopy were 2687 +/- 30 and 2758 +/- 30 Da, respectively, indicating that (i) the only difference between alpha and beta was the presence of the N-terminal alanine residue in beta, and that (ii) in addition to the sequenced residues, two to three unidentified amino acid residues are present at the C-terminal ends of the alpha and beta peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcifying epithelial odontogenic (Pindborg) tumor-associated amyloid consists of a novel human protein

Calcifying epithelial odontogenic tumors (CEOTs), also known as Pindborg tumors, are characterized by the presence of squamous-cell proliferation, calcification, and, notably, amyloid deposits. On the basis of immunohistochemical analyses, the amyloidogenic component had heretofore been deemed to consist of cytokeratin-related or other molecules; however, its chemical composition had never been elucidated. We have used our microanalytic techniques to characterize the protein nature of CEOT-associated amyloid isolated from specimens obtained from 3 patients. As evidenced by the results of amino-acid sequencing and mass spectrometry, the fibrils were found to be composed of a polypeptide of approximately 46 mer. This component was identical in sequence to the N-terminal portion of a hypothetical 153-residue protein encoded by the FLJ20513 gene cloned from the human KATO III cell line. That the amyloid protein was derived from this larger molecule was demonstrated by reverse transcription-polymerase chain reaction amplification of tumor-cell RNA where a full-length FLJ20513 transcript was found. Furthermore, immunohistochemical analyses revealed that the amyloid within the CEOTs immunostained with antibodies prepared against a synthetic FLJ20513-related dodecapeptide. Our studies provide unequivocal evidence that CEOT-associated amyloid consists of a unique and previously undescribed protein that we provisionally designate APin.