Anders H. Johnsen

A Family of Insulin-Like Growth Factor II mRNA-Binding Proteins Represses Translation in Late Development

ABSTRACT Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5′ untranslated regions (5′ UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins (IMPs) that exhibit multiple attachments to the 5′ UTR from the translationally regulated IGF-II leader 3 mRNA but are unable to bind to the 5′ UTR from the constitutively translated IGF-II leader 4 mRNA. IMPs contain the unique combination of two RNA recognition motifs and four hnRNP K homology domains and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growth-dependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 –luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5′ UTR-binding proteins control IGF-II biosynthesis during late mammalian development.

Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung

The leading cause of mortality in patients with cystic fibrosis (CF) is respiratory failure due in large part to chronic lung infection with Pseudomonas aeruginosa strains that undergo mucoid conversion, display a biofilm mode of growth in vivo and resist the infiltration of polymorphonuclear leukocytes (PMNs), which release free oxygen radicals such as H2O2. The mucoid phenotype among the strains infecting CF patients indicates overproduction of a linear polysaccharide called alginate. To mimic the inflammatory environment of the CF lung, P. aeruginosa PAO1, a typical non-mucoid strain, was grown in a biofilm. This was treated with low levels of H2O2, as if released by the PMNs, and the formation of mucoid variants was observed. These mucoid variants had mutations in mucA, which encodes an anti-sigma factor; this leads to the deregulation of an alternative sigma factor (sigma22, AlgT or AlgU) required for expression of the alginate biosynthetic operon. All of the mucoid variants tested showed the same mutation, the mucA22 allele, a common allele seen in CF isolates. The mucoid mucA22 variants, when compared to the smooth parent strain PA01, (i) produced 2-6-fold higher levels of alginate, (ii) exhibited no detectable differences in growth rate, (iii) showed an unaltered LPS profile, (iv) were approximately 72% reduced in the amount of inducible-beta-lactamase and (v) secreted little or no LasA protease and only showed 44% elastase activity. A characteristic approximately 54 kDa protein associated with alginate overproducing strains was identified as AlgE (Alg76) by N-terminal sequence analysis. Thus, the common phenotype of the mucoid variants, which included a genetically engineered mucA22 mutant, suggested that the only mutation incurred as a result of H2O2 treatment was in mucA. When a P. aeruginosa biofilm was repeatedly exposed to activated PMNs in vitro, mucoid variants were also observed, mimicking in vivo observations. Thus, PMNs and their oxygen by-products may cause P. aeruginosa to undergo the typical adaptation to the intractable mu- coid form in the CF lung. These findings indicate that gene activation in bacteria by toxic oxygen radicals, similar to that found in plants and mammalian cells, may serve as a defence mechanism for the bacteria. This suggests that mucoid conversion is a response to oxygen radical exposure and that this response is a mechanism of defence by the bacteria. This is the first report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form of P. aeruginosa in the CF lung. These findings may provide a basis for the development of anti-oxidant and anti-inflammatory therapy for the early stages of infection in CF patients.

hCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules

A 19 kDa protein was identified in specific granules of human neutrophils. A full‐length cDNA clone was isolated from a human CML cDNA library, based on amino‐acid sequences of isolated tryptic fragments. This clone includes the recently identified cDNA for FALL‐39/CAP‐18. Aminoacid sequences of proteolytic fragments derived both from the conserved N‐terminal cathelin‐like region and the highly variable C‐terminal region characteristic of this family of bactericidal, LPS binding proteins, were in complete agreement with the sequence deduced from the cDNA. Thus, the 19 kDa protein is hCAP‐18, stored as a ‘pro‐peptide’ in specific granules.

Structure, measurement, and secretion of human glucagon-like peptide-2

By using radioimmunoassays toward the cDNA-predicted amino acid sequence of human glucagon-like peptide-2, a peptide was isolated from extracts of human ileum. By mass spectrometry and Edman sequencing, this peptide was identified as human proglucagon 126-158. High-performance liquid chromatography analyses indicated that a similar immunoreactive peptide (iGLP-2) was present in human plasma. Human plasma concentrations of iGLP-2 were elevated 3- to 4-fold at 1 to 2 h after ingestion of 800 to 1200 kcal meals.

Molecular Composition of IMP1 Ribonucleoprotein Granules

Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100–300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly(A)-binding proteins, and mRNAs. Moreover granules contained CBP80 and factors belonging to the exon junction complex and lacked eIF4E, eIF4G, and 60 S ribosomal subunits, indicating that embodied mRNAs are not translated. Granules embodied mRNAs corresponding to about 3% of the human embryonic kidney 293 mRNA transcriptome. Messenger RNAs encoding proteins participating in the secretory pathway and endoplasmic reticulum-associated quality control, as well as ubiquitin-dependent metabolism, were enriched in the granules, reinforcing the concept of RNP granules as post-transcriptional operons.

Allatostatic neuropeptides from the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae). Identification, immunolocalization and activity

Four allatostatic neuropeptides were isolated from extracts of the brain of the cockroach Blattella germanica. The primary structures of these peptides were assigned as Leu-Tyr-Asp-Phe-Gly-Leu-NH2 (BLAST-1), Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (BLAST-2), Ala-Gly-Ser-Asp-Gly-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 (BLAST-3) and Ala-Pro-Ser-Ser-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 (BLAST-4). Each of the peptides showed C-terminal amino acid sequence similarity to cockroach allatostatins and blowfly callatostatins. The four peptides inhibited in vitro juvenile hormone production by corpora allata from virgin females of B. germanica. Immunoreactivity against allatostatins was seen in the lateral neurosecretory neurons and in the axonal pathway leading to the corpora allata.

Human neutrophil gelatinase: A marker for circulating blood neutrophils. Purification and quantitation by enzyme linked immunosorbent assay

Abstract:  Human neutrophil gelatinase was purified to apparent homogeneity. The N‐terminal amino‐acid sequence of the purified enzyme could be aligned to an internal part of the cDNA‐derived amino‐acid sequence of 92‐kDa type IV collagenase from SV 40‐transfected human lung fibroblasts and from a TPA differentiated monocytic cell line, U937. Total amino‐acid composition of U937 and neutrophil gelatinases was identical. Gelatinase was susceptible to treatment with o‐ and n‐glycanase, indicating that posttranslational addition of oligosaccharide side chains occurs. An enzyme‐linked immunosorbent assay for gelatinase was developed using specific polyclonal rabbit antibodies. The assay was specific, sensitive, accurate, and reproducible. Ninety percent range for plasma gelatinase from normal subjects was 17.3 to 102.9 ng/ml. In patients treated with cytostatic agents for non‐Hodgkins lymphoma, there was a parallel drop in plasma gelatinase and peripheral granulocyte count. This indicates that plasma gelatinase is a marker for circulating neutrophils. Plasma gelatinase does not seem to reflect bone marrow cellularity.

The Human Antibacterial Cathelicidin, hCAP-18, Is Bound to Lipoproteins in Plasma

Cathelicidins are a family of antibacterial and lipopolysaccharide-binding proteins. hCAP-18, the only human cathelicidin, is a major protein of the specific granules of human neutrophils. The plasma level of hCAP-18 is >20-fold higher than that of other specific granule proteins relative to their levels within circulating neutrophils. The aim of this study was to elucidate the background for this high plasma level of hCAP-18. Plasma was subjected to molecular sieve chromatography, and hCAP-18 was found in distinct high molecular mass fractions that coeluted with apolipoproteins A-I and B, respectively. The association of hCAP-18 with lipoproteins was validated by the cofractionation of hCAP-18 with lipoproteins using two different methods for isolation of lipoproteins from plasma. Furthermore, the level of hCAP-18 in delipidated plasma was <1% of that in normal plasma. Immunoprecipitation of very low, low, and high density lipoprotein particles with anti-apolipoprotein antibodies resulted in coprecipitation of hCAP-18. The binding of hCAP-18 to lipoproteins was mediated by the antibacterial C-terminal part of the protein. The binding of hCAP-18 to lipoproteins suggests that lipoproteins may play an important role as a reservoir of this antimicrobial protein.

Human galanin: Primary structure and indentification of two molecular forms

From acid/ethanol extracts of surgical specimens of human large intestine we isolated two peptides, in approximately equal amounts, that reacted with an antiserum against porcine galanin. By amino acid analysis, sequence analysis and mass spectrometry, the larger of the two peptides was found to consist of 30 amino acid residues, the sequence of which was identical to that of porcine galanin except for the following substitutions: Val16, Asn17, Asn36, Thr20 and Scr30. Unlike porcine galanin, the caboxy‐terminus was not amidated. The smaller peptide corresponded to the first 19 amino acid residues counted from the N‐terminus of the 30 residue peptide (again without amidation). The structural analysis was repeated on another batch of tissue with identical results. By HPLC analysis of extracts of specimens from a further 4 patients, the same peptides were identified. Thus, human galanin includes two peptides of 19 and 30 amino acids that share the sequence of the N‐terminal 15 residues with other mammalian galanins, but exhibit characteristic differences in the remaining part of the molecules.

The Ubx2 and Ubx3 Cofactors Direct Cdc48 Activity to Proteolytic and Nonproteolytic Ubiquitin-Dependent Processes

Valosin-containing protein, VCP/p97 or Cdc48, is a eukaryotic ATPase involved in membrane fusion, protein transport, and protein degradation. We describe two proteins, Ubx2 and Ubx3, which interact with Cdc48 in fission yeast. Ubx3 is the ortholog of p47/Shp1, a previously described Cdc48 cofactor involved in membrane fusion, whereas Ubx2 is a novel protein. Cdc48 binds the UBX domains present in both Ubx2 and Ubx3, indicating that this domain is a general Cdc48-interacting module. Ubx2 and Ubx3 also interact with ubiquitin chains. Disruption of the ubx3(+)-gene causes both temperature and canavanine sensitivity and stabilizes some ubiquitin-protein conjugates including the CDK inhibitor Rum1, but not a model substrate of the ER-degradation pathway. Moreover the ubx3 null displays synthetic lethality with a pus1 null mutant, a multiubiquitin binding subunit of the 26S proteasome. In contrast, the ubx2 null mutant did not display any obvious protein-degradation phenotype. In conclusion Ubx3/p47 is not, as previously thought, only important for membrane fusion; its also important for the specific degradation of a subset of cell proteins. Our genetic analyses revealed that Ubx3/p47 functionally parallels a substrate receptor of the 26S proteasome, Pus1/Rpn10, indicating that the Cdc48-Ubx3 complex is involved in delivering substrates to the 26S proteasome.