Biomedical subjects

Anders H Johnsen

Publications and source records attributed to Anders H Johnsen.

At least 19 recordsLinked to original sources

PubMed2006

Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor.

We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon. Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre-existing 26 S proteasomes in cell extracts. Adrm1 may be distantly related to the yeast proteasome subunit Rpn13, mutants of which are reported to display no obvious phenotype. Accordingly, knock-down of Adrm1 in HeLa cells had no effect on the amount of proteasomes, or on degradation of bulk cell protein, or accumulation of polyubiquitinylated proteins. This indicates that Adrm1 has a specialised role in proteasome function.

Amino Acid Sequence↗

PubMed2006

Isolation and characterization of human apolipoprotein M-containing lipoproteins.

Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoM-containing lipoproteins were predominantly of HDL size; approximately 5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, paraoxonase 1, and apoB. ApoM-containing HDL (HDL(apoM+)) contained significantly more free cholesterol than HDL lacking apoM (HDL(apoM-)) (5.9 +/- 0.7% vs. 3.2 +/- 0.5%; P < 0.005) and was heterogeneous in size with both small and large particles. HDL(apoM+) inhibited Cu(2+)-induced oxidation of LDL and stimulated cholesterol efflux from THP-1 foam cells more efficiently than HDL(apoM-). In conclusion, our results suggest that apoM is associated with a small heterogeneous subpopulation of HDL particles. Nevertheless, apoM designates a subpopulation of HDL that protects LDL against oxidation and stimulates cholesterol efflux more efficiently than HDL lacking apoM.

Apolipoproteins↗

PubMed2006

ADAM12 is a four-leafed clover: the excised prodomain remains bound to the mature enzyme.

The ADAMs (a disintegrin and metalloprotease) comprise a family of multidomain proteins with metalloprotease, cell adhesion, and signaling activities. Human ADAM12, which is implicated in diseases such as cancer, is expressed in two splice forms, the transmembrane ADAM12-L and the shorter and soluble ADAM12-S. ADAM12 is synthesized as a zymogen with the prodomain keeping the metalloprotease inactive through a cysteine-switch mechanism. Maturation and activation of the protease involves the cleavage of the prodomain in the trans-Golgi or possibly at the cell surface by a furin-peptidase. The aim of the present study was to determine the fate of the prodomain following furin cleavage. Here we demonstrate that, following cleavage of the human ADAM12-S prodomain in the trans-Golgi by a furin-peptidase, the prodomain remains non-covalently associated with the mature molecule. Accordingly, both the 68-kDa mature form of ADAM12-S and the 25-kDa prodomain could be detected using domain-specific antisera in immunoprecipitation and Western blot analyses of human serum ADAM12 and purified recombinant human ADAM12. Using electron microscopy after negative staining we have furthermore obtained the first visualization of a full-length ADAM molecule, human ADAM12-S, and report that it appears to be a compact clover composed of four globular domains, one of which is the prodomain. Finally, our data demonstrate that the presence of the metalloprotease domain appears to be sufficient for the prodomain to remain associated with the mature ADAM12-S. Thus, we conclude that the prodomain of human ADAM12-S is an integral domain of the mature molecule and as such might have specific biological functions in the extracellular space.

ADAM Proteins↗

PubMed2006

Identification and characterization of glucoamylase from the fungus Thermomyces lanuginosus.

The glucoamylase from the thermophilic fungus Thermomyces lanuginosus has a molecular weight of 66 kDa and was characterized with isoelectric point, pH and temperature optimum of 3.8-4.0, 5.0 and 70 degrees C, respectively. In addition, the activation energy is 60.4 kJ/mol, Km is 3.5 mM and kcat is 25.3 s(-1). The glucoamylase was partially sequenced on the protein level, and the complete glucoamylase gene including its promoter (but excluding its terminator region) was cloned and sequenced. The glucoamylase protein comprises 617 amino acid residues and shows 60% identity with the glucoamylase from the thermophilic fungus Talaromyces emersonii. cDNA encoding Thermomyces lanuginosus glucoamylase was expression cloned into Pichia pastoris, producing approximately 7.4 U/ml. It was concluded that alternative mRNA splicing as it might occur in Aspergillus niger glucoamylase is not responsible for the occurrence of different glucoamylase isoforms in Thermomyces lanuginosus.

Ascomycota↗

PubMed2005

beta-Microseminoprotein binds CRISP-3 in human seminal plasma.

beta-Microseminoprotein (MSP) and cysteine-rich secretory protein 3 (CRISP-3) are abundant constituents of human seminal plasma. Immunoprecipitation and gel filtration of seminal plasma proteins combined with examination of the proteins in their pure form showed that MSP and CRISP-3 form stable, non-covalent complexes. CRISP-3 binds MSP with very high affinity, as evidenced by surface plasmon resonance. Due to far higher abundance of MSP in prostatic fluid, it manifests large overcapacity for CRISP-3 binding. Structural similarity with an MSP-binding protein from blood plasma suggests that CRISP-3 binds MSP through its aminoterminal SCP-domain.

Humans↗

PubMed2005

Prodefensins are matrix proteins of specific granules in human neutrophils.

Defensins are potent antimicrobial and proinflammatory peptides. The human neutrophil defensins human neutrophil peptide (HNP)-1-3 are synthesized as 94 amino acide (aa) preproHNPs, which are converted to 75 aa proHNPs by cotranslational removal of a 19 aa endoplasmic reticulum signal peptide. At the promyelocytic stage of myelopoiesis, proHNPs are further proteolytically modified and accumulate in azurophil granules as 29-30 aa HNPs. In contrast, proHNPs produced by more mature myeloid cells are not subjected to proteolytic cleavage and undergo a high degree of constitutive exocytosis. The proHNPs are devoid of antimicrobial potential, and the significance of their secretion is unknown. To investigate whether mature neutrophils contain proHNPs, we developed antibodies against proHNP-1 by DNA immunization of rabbits. In addition, antibodies against the 45 aa proHNP pro-piece were raised by conventional immunization procedures. These antibodies allowed detection of proHNPs in homogenates of peripheral blood neutrophils. The proHNPs were isolated by affinity chromatography, and their identity was confirmed by mass spectrometry and N-terminal aa sequence analysis. Finally, the neutrophil proHNPs were identified as novel matrix proteins of specific granules by subcellular fractionation experiments, release studies, and immunoelectron microscopy. Thus, human neutrophils not only store large amounts of mature defensins in azurophil granules but also contain a more easily mobilized reservoir of unprocessed prodefensins in specific granules.

Antibodies↗

PubMed2004

Processing of chicken progastrin at post-Phe bonds by an aspartyl protease.

Prohormones mature to biologically active peptide hormones through posttranslational modifications, which include endoproteolytic cleavages. Cleavages at mono- and dibasic sites are well characterized, and several of the responsible prohormone convertases have been identified. There is, however, evidence that endoproteolytic maturation occurs also at other sites. Among these, post-Phe cleavage occurs in the maturation of chicken progastrin, where the processing to gastrin-30 has been examined in detail. In this study we have characterized an endoprotease of the aspartic acid protease family in chicken and human tissue capable of cleaving at the Phe site. Enzymatic activity was monitored by radioimmunoassays using antibodies specific for the N- and C-termini exposed after cleavage. Analysis showed that only pepstatin, a specific inhibitor of aspartic proteases, inhibited the enzyme. The pH optimum of the enzyme ranged from pH 2 to pH 5. Amino acid substitution from Phe to Ala in the substrate completely abolished enzyme activity. The endoproteolytic activity was identified in chicken antrum and pectoral muscle as well as human cardiac and prostate extracts, suggesting that the enzyme has widespread biological functions. Experiments using recombinant cathepsin D and E indicated that neither is responsible for the endoproteolytic cleavage of chicken progastrin at post-Phe bonds.

Amino Acid Sequence↗

PubMed2004

Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma.

Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin-like substances found in lizard saliva or snake venom. Human CRISP-3 is present in exocrine secretions and in secretory granules of neutrophilic granulocytes and is believed to play a role in innate immunity. On the basis of the relatively high content of CRISP-3 in human plasma and the small size of the protein (28 kDa), we hypothesized that CRISP-3 in plasma was bound to another component. This was supported by size-exclusion chromatography and immunoprecipitation of plasma proteins. The binding partner was identified by mass spectrometry as alpha(1)B-glycoprotein (A1BG), which is a known plasma protein of unknown function and a member of the immunoglobulin superfamily. We demonstrate that CRISP-3 is a specific and high-affinity ligand of A1BG with a dissociation constant in the nanomolar range as evidenced by surface plasmon resonance. The A1BG-CRISP-3 complex is noncovalent with a 1:1 stoichiometry and is held together by strong electrostatic forces. Similar complexes have been described between toxins from snake venom and A1BG-like plasma proteins from opossum species. In these cases, complex formation inhibits the toxic effect of snake venom metalloproteinases or myotoxins and protects the animal from envenomation. We suggest that the A1BG-CRISP-3 complex displays a similar function in protecting the circulation from a potentially harmful effect of free CRISP-3.

Blood Proteins↗

PubMed2004

Naming progastrin-derived peptides.

The antral hormone gastrin continues to be in focus, because its hormonal and growth promoting effects are essential both for the function of the normal stomach and for the pathogenesis of major dyspeptic and neoplastic diseases. Deduction of the progastrin structure has improved the insight in the cellular synthesis of gastrin, but has also revealed that the biosynthetic machinery is complex, and, accordingly, that progastrin is processed to a multitude of more or less bioactive fragments. The naming of these fragments has, however, become inconsistent and confusing. Therefore, we propose a systematic nomenclature for progastrin-derived peptides of which there are three classes: (I) The gastrins with the evolutionary preserved tetrapeptide amide (Trp-Met-Asp-PheNH2) at the C-terminus, which ensures high-affinity binding to the gastrin (CCK-B) receptor. Among the gastrins, gastrin-34 and gastrin-17 constitute the primary forms. (II) Processing intermediates, which are early products of progastrin that contain the structure of the primary gastrins within their sequence, but still cannot bind the gastrin receptor due to insufficient processing at their C-terminus. (III) Flanking fragments from the N- and C-termini of progastrin that do not contain any primary gastrin in their sequence, but nevertheless may undergo posttranslational processing. Each fragment can be specified with suffixes corresponding to the derived sequence in progastrin.

Amino Acid Sequence↗

PubMed2004

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; it's 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.

Adenosine Triphosphatases↗

PubMed2004

Expression of actin and tubulins in purified human basophil leukocytes upon stimulation with IL-3.

BACKGROUND: Pharmacological and morphological results indicate that the cytoskeleton proteins tubulins and actin play a role in the histamine release process in basophil leukocytes and mast cells. In this report, we investigate the expression of these cytoskeleton proteins in purified human basophils upon stimulation with anti-IgE and IL-3. METHODS: Human basophils were purified using a negative selection procedure. They were incubated for 6 h with anti-IgE and/or IL-3 in radiolabeling media. Proteins in the cells were analyzed by two-dimensional gel electrophoresis and autoradiography. Actin and alpha- and beta-tubulin were identified by amino acid sequence analysis or matrix assisted laser desorption/ionisation time-of-flight mass spectrometry. The synthesis of these proteins under different experimental conditions was evaluated by densitometry. RESULTS: Actin is regulated dose dependently by IL-3 with an optimum concentration of 100 ng/ml, corresponding to the optimal IL-3 concentration for enhancement of histamine release. The synthesis of tubulins was not significantly upregulated by IL-3 or anti-IgE alone, but there was a slight and significant upregulation of tubulins upon stimulation with both IL-3 and anti-IgE in doses optimal for maximal histamine release. CONCLUSIONS: The de novo synthesis of actin is regulated dose dependently by IL-3 in purified human basophils in short-term culture. The optimum IL-3 concentration is the same as for enhancement of histamine release, suggesting that the same processes that regulate IL-3-induced enhancement of histamine release also regulate the expression of actin. The finding that the expression of the tubulins is not upregulated by IL-3 alone suggests that regulation of tubulin synthesis is not a pathway by which IL-3 in itself primes basophil histamine release.

Actins↗

PubMed2004

Enhanced peptide secretion by gene disruption of CYM1, a novel protease in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a widely used host in the production of therapeutic peptides and proteins. Here we report the identification of a novel endoprotease in S. cerevisiae. It is encoded by the CYM1 gene and is specific for the C-terminus of basic residues of heterologously expressed peptides. Gene disruption of CYM1 not only reduced the intracellular proteolysis, but also enhanced the secretion of heterologously expressed peptides such as growth hormone, pro-B-type natriuretic peptide and pro-cholecystokinin. Cym1p resembles metalloendoproteases of the pitrilysin family with the HXXEH(X)E(71-77) catalytic domain as seen in insulysin, nardilysin and human metalloprotease 1. It is a nuclear encoded protease that localizes to mitochondria without a hydrophobic N-terminal signal sequence or a C-terminal tail-anchor. The protease does not require post-translational processing prior to activation and it contains cytosolic activity that processes peptides designated for the secretory pathway prior to translocation into the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed2003

Processing of seminal plasma hCAP-18 to ALL-38 by gastricsin: a novel mechanism of generating antimicrobial peptides in vagina.

The human cathelicidin, hCAP-18, is expressed both in neutrophils and in epithelial cells. hCAP-18 is processed to the antimicrobial peptide LL-37 by proteinase 3 in neutrophils. hCAP-18 is highly expressed in the epididymis with a subsequent high concentration in seminal plasma where the protein is present in its unprocessed and antimicrobially inactive form. We report here that hCAP-18 in seminal plasma is processed to generate a 38-amino acid antimicrobial peptide ALL-38 by the prostate-derived protease gastricsin when incubated at a pH corresponding to the vaginal pH. In accordance with this, seminal plasma derived hCAP-18 was found in its processed form in the vagina following sexual intercourse. The antimicrobial activity of ALL-38 against a variety of microorganisms tested is equal to that of LL-37. This enzymatic activation of a proantimicrobial substance in seminal plasma following exposure to the vaginal milieu represents a novel mechanism to prevent infection following sexual intercourse.

Amino Acid Sequence↗

PubMed2003

Purification and characterization of osteopontin from human milk.

Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further separated into its different molecular forms by means of a two-step procedure, involving size exclusion chromatography and reverse phase chromatography. A rabbit polyclonal antibody was raised to purified intact OPN and high M(r) OPN components; the immunoreactivity of both forms was almost equal when investigated by enzyme immunoassay (EIA). The procedures facilitate the purification of intact OPN and OPN fragments for purposes of standardization, preparation of monospecific antibodies, and functional studies.

Antibodies↗

PubMed2003

Tissue expression of copines and isolation of copines I and III from the cytosol of human neutrophils.

Copines are a recently identified group of proteins characterized by two Ca(2+)-binding C2-domains at the N terminus and an A-domain at the C terminus. Although pEST sequences indicate the existence of at least seven copines in man, only copines I, III, and VI have been identified at protein level. Here, we describe the isolation of copines I and III in the cytosol of human neutrophils by use of Ca(2+)-induced hydrophobic chromatography. This is the first demonstration that copines are coexpressed in the same cell. We found that copine III exists in the cytosol of human neutrophils as a monomer with a blocked N terminus. Copines I and III undergo conformational changes upon Ca(2+) binding that lead to exposure of hydrophobic patches. Examination of RNA from 68 human tissues demonstrated that copines I-III are ubiquitously expressed whereas copines IV-VII each has a more restricted and individual expression profile. Expression of copines I-III was also demonstrated in neutrophil precursors from bone marrow. Copine I was uniformly expressed at all stages of neutrophil differentiation, whereas copine II and even more so, copine III were expressed in the more immature neutrophil precursors, which indicates an individual function of these copines.

Amino Acid Sequence↗

PubMed2002

Defensin-rich granules of human neutrophils: characterization of secretory properties.

The various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related alpha-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil alpha-defensins HNP 1-3. This allowed us to quantify the exocytosis of alpha-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of alpha-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release.

Antibodies↗

PubMed2002

An ELISA for SGP28/CRISP-3, a cysteine-rich secretory protein in human neutrophils, plasma, and exocrine secretions.

Specific granule protein of 28 kDa (SGP28), also termed cysteine-rich secretory protein 3 (CRISP-3), is a glycoprotein that belongs to a family of cysteine-rich secretory proteins (CRISPs). SGP28 was originally discovered in human neutrophils, but transcripts are widely distributed in exocrine glands (salivary glands, pancreas, and prostate) and also found at lower levels in epididymis, ovary, thymus, and colon. The function of SGP28/CRISP-3 is not yet known. Similarities to pathogenesis-related proteins in plants and the expression in neutrophils and exocrine glands suggest that SGP28/CRISP-3 may play a role in innate host defense. We describe here the production of a recombinant, C-terminally truncated form of CRISP-3 (rCRISP-3Delta) and the generation of polyclonal antibodies against rCRISP-3Delta that are useful in immunoblotting and immunocytochemistry. We present a specific, accurate, and reproducible enzyme-linked immunosorbant assay (ELISA) for the measurement of CRISP-3 with a detection limit of 2 ng/ml. We further demonstrate the presence of CRISP-3 protein in human plasma (6.3 microg/ml), saliva (21.8 microg/ml), seminal plasma (11.2 microg/ml), and sweat (0.15 microg/ml), and describe the coexistence of two different molecular weight forms of CRISP-3, representing an N-glycosylated and a non-glycosylated form of the mature protein.

Amidohydrolases↗

PubMed2002

The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy.

The hypothesis that chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, may be a precursor of biologically active peptides, rests on observed activities of peptide fragments largely produced by exogenous protease digestion of the bovine protein. Here we have adopted a modified proteomic strategy to isolate and characterise human CgA-derived peptides produced by endogenous prohormone convertases. Initial focus was on an insulinoma as previous studies have shown that CgA is rapidly processed in pancreatic beta cells and that tumours arising from these express appropriate prohormone convertases. Eleven novel peptides were identified arising from processing at both monobasic and dibasic sites and processing was most evident in the C-terminal domain of the protein. Some of these peptides were identified in endocrine tumours, such as mid-gut carcinoid and phaeochromocytoma, which arise from endocrine cells of different phenotype and in different anatomical sites. Two of the most interesting peptides, GR-44 and ER-37, representing the C-terminal region of CgA, were found to be amidated. These data would imply that the intact protein is C-terminally amidated and that these peptides are probably biologically active. The spectrum of novel CgA-derived peptides, described in the present study, should provide a basis for biological evaluation of authentic entities.

Amino Acid Sequence↗