Biomedical subjects

H R Rackwitz

Publications and source records attributed to H R Rackwitz.

At least 19 recordsLinked to original sources

PubMed2001

Characterization of peptide substrates and viral enzyme that affect the cleavage site specificity of the human spumaretrovirus proteinase.

Oligopeptides that correspond to proteolytic cleavage site junctions of the native Gag and Pol proteins are specifically cleaved by retroviral aspartate proteases (PRs). The role of the flap subdomain of the PR of the human spumaretrovirus (HSRV) and of substrate peptides in cleavage site specificity was analyzed by site-directed mutagenesis. Native and mutant peptides were subjected to proteolysis by the authentic and mutated recombinant viral enzyme. The results reveal that Glu residue 54 of the HSRV PR is an essential specificity determinant for proteolytic processing of the structural proteins. Peptides that represent in vivo cleavage sites were susceptible to proteolysis by the recombinant HSRV PR, but one peptide located at the junction between the PR and reverse transcriptase domains was completely resistant to cleavage. Thus the data indicate that a proteolytic cleavage between these domains does not occur in vivo. Naturally occurring and mutant forms of the cleavage-resistant peptide were therefore analyzed by circular dichroism to determine if differences existed in the secondary structures of the peptides that did or did not serve as substrates. The data show that differences in the secondary structure of the native and mutant peptides analyzed does not seem to play a crucial role for cleavage site specificity in HSRV PR. Instead highly conserved hydrophobic residues at distinct positions of the HSRV cleavage site junctions contribute to the specificity observed as reported for HIV-1 PR.

Amino Acid Sequence↗

PubMed2001

Activation of pro-astacin. Immunological and model peptide studies on the processing of immature astacin, a zinc-endopeptidase from the crayfish Astacus astacus.

To contribute knowledge of the processing and activation of invertebrate proteolytic enzymes, we studied the metalloprotease astacin, a digestive enzyme from the freshwater crayfish Astacus astacus (decapod crustacean). It is the prototype of the protein family of astacins, members of which occur in organisms from bacteria to man and are involved in a variety of physiological reactions. According to its genomic structure, astacin is produced as a zymogen [Geier, G., Jacob, E., Stöcker, W. & Zwilling, R. (1997) Arch. Biochem. Biophys. 337, 300-307]. To localize and follow the processing of pro-astacin in different parts of the digestive tract, we synthesized two peptides covering the pro part of pro-astacin and raised antibodies against them. In addition, antiserum against the whole active astacin was produced. Using immunohistochemical investigation, we detected pro-astacin in the F cells of the hepatopancreas and all the way into the tubular lumen and the collecting ducts of this gland. Immunoblot assays revealed only active astacin, and never pro-astacin, present in the cardiac stomach. We conclude from these studies that astacin is secreted into the lumen of the hepatopancreatic tubules in its pro form and is activated on its way to the stomach. To investigate which of the two endopeptidases found in the digestive tract of crayfish, astacin or trypsin, is responsible for cleaving the propeptide from pro-astacin, we synthesized different peptides that mimick the activation site. MS analysis of the cleavage products of astacin and trypsin showed that astacin is capable of catalyzing its own activation. Any contribution of trypsin would require the successive action of an aminopeptidase. Substituting glycine for arginine at position -1 of the activation site does not prevent astacin activity. As most members of the astacin protein family have basic amino-acid residues in this position, in these cases also astacin-specific cleavage would be possible.

Amino Acid Sequence↗

PubMed2000

Nuclear coactivator protein p100 is present in endoplasmic reticulum and lipid droplets of milk secreting cells.

We have identified the p100 protein, previously known as a novel cellular coactivator, as a constituent of endoplasmic reticulum and cytosolic lipid droplets from milk secreting cells. Cytosolic lipid droplets of terminally differentiated mammary epithelial cells are secreted as milk lipid globules. However, milk lipid globules did not have detectable amounts of p100 protein. The p100 protein was found also in cytosol from lactating mammary gland, in storage lipid droplets from mouse adipocytes, and in endoplasmic reticulum from liver. Immunofluorescence microscopy of mammary epithelial cells confirmed the presence of p100 in non-nuclear regions of these cells. Partial sequence analysis of tryptic peptides from p100 from cow mammary gland showed extensive homology with the reported sequence of p100 determined from a human cDNA. Antibodies against a peptide synthesized to duplicate a sequence in human p100 recognized a protein of the size of p100 in cow, mouse and rat cell fractions.

Adipocytes↗

PubMed1999

The catalog of human hair keratins. I. Expression of the nine type I members in the hair follicle.

The human type I hair keratin subfamily comprises nine individual members, which can be subdivided into three groups. Group A (hHa1, hHa3-I, hHa3-II, hHa4) and B (hHa7, hHa8) each contains structurally related hair keratins, whereas group C members hHa2, hHa5, and hHa6 represent structurally rather unrelated hair keratins. Antibodies produced against these individual hair keratins, first analyzed for specificity by one- dimensional Western blots of total hair keratins, were used to establish the two-dimensional catalog of the human type I hair keratin subfamily. The catalog comprises two different series of type I hair keratins: a strongly expressed, Coomassie-stainable series containing hair keratins hHa1, hHa3-I/II, hHa4, and hHa5, and a weakly expressed, immunodetectable series harboring hHa2, hHa6 hHa7, and hHa8. In situ hybridization and immunohistochemical expression studies on scalp follicles show that two hair keratins, hHa2 and hHa5, define the early stage of hair differentiation, i.e. hHa5 expression in hair matrix and hHa5/hHa2 coexpression in the early hair cuticle cells. Whereas cuticular differentiation proceeds without the expression of further type I hair keratins, matrix cells embark on the cortical pathway by sequentially expressing hHa1, hHa3-I/II, and hHa4, which are supplemented by hHa6 at an advanced stage of cortical differentiation, and hHa8, which is expressed heterogeneously in cortex cells. Thus, six type I hair keratins are involved in the terminal differentiation of anagen hairs. The expression of hHa7 is conspicuously different from that of the other hair keratins in that it does not occur in the large anagen follicles of terminal scalp hairs but only in central cortex cells of the rare and small follicle type that gives rise to vellus hairs.

Blotting, Western↗

PubMed1999

Plakophilin 3--a novel cell-type-specific desmosomal plaque protein.

Desomosomes are cell-cell adhesion structures of epithelia and some non-epithelial tissues, such as heart muscle and the dendritic reticulum of lymph node follicles, which on their cytoplasmic side anchor intermediate filaments at the plasma membrane. Besides clusters of specific transmembrane glycoproteins of the cadherin family (desmogleins and desmocollins), they contain several desmosomal plaque proteins, such as desmoplakins, plakoglobin, and one or more plakophilins. Using recombinant DNA and immunological techniques, we have identified a novel desmosomal plaque protein that is closely related to plakophilins 1 and 2, both members of the "armadillo-repeat" multigene family, and have named it plakophilin 3 (PKP3). The product of the complete human cDNA defines a protein of 797 amino acids, with a calculated molecular weight of 87.081 kDa and an isoelectric point of pH 10.1. Northern blot analysis has shown that PKP3 mRNA has a size of approximately 2.9 kb and is detectable in the total RNA of cells of stratified and single-layered epithelia. With the help of specific poly- and monoclonal antibodies we have localized PKP3, by immunofluorescence or immunoelectron microscopy, to desmosomes of most simple and almost all stratified epithelia and cell lines derived therefrom, with the remarkable exception of hepatocytes and hepatocellular carcinoma cells. We have also determined the structure of the human PKP3 gene and compared it with that of plakophilin 1 (PKP1). Using fluorescence in situ hybridization, we have localized the human genes for the three known plakophilins to the chromosomes 1q32 (PKP1), 12p11 (PKP2) and 11p15 (PKP3). The similarities and differences of the diverse plakophilins are discussed.

Amino Acid Sequence↗

PubMed1999

Molecular characterization of proteolytic processing of the Gag proteins of human spumavirus.

Spumaviruses, or foamy viruses, express Gag proteins that are incompletely processed by the viral protease in cell cultures. To delineate the proteolytic cleavage sites between potential Gag subdomains, recombinant human spumaretrovirus (HSRV) Gag proteins of different lengths were expressed, purified by affinity chromatography, and subjected to HSRV protease assays. HSRV-specific proteolytic cleavage products were isolated and characterized by Western blotting. Peptides spanning potential cleavage sites, as deduced from the sizes of the proteolytic cleavage products, were chemically synthesized and assayed with HSRV protease. The cleaved peptides were then subjected to mass spectrometry. In control experiments, HSRV protease-deficient mutant proteins were used to rule out unspecific processing by nonviral proteases. The cleavage site junctions identified and the calculated sizes of the cleavage products were in agreement with those of the authentic cleavage products of the HSRV Gag proteins detectable in viral proteins from purified HSRV particles and in virus-infected cells. The biological significance of the data was confirmed by mutational analysis of the cleavage sites in a recombinant Gag protein and in the context of the infectious HSRV DNA provirus.

Animals↗

PubMed1998

Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases.

We report the human DNA and protein sequence of adipophilin and its association with the surface of lipid droplets. The amino acid sequence of human adipophilin has been determined by using cDNA clones from several tissues and confirmed by the reverse transcription/polymerase chain reaction method and Edman sequencing. The open reading frame of adipophilin encodes a polypeptide with a calculated molecular weight of 48.1 kDa and an isoelectric point of 6.72. By immunofluorescence and electron-microscopic localization with newly raised specific poly- and monoclonal antibodies, we show that this protein is not restricted to adipocytes as previously indicated by studies of the mouse homologous protein, adipose-differentiation-related protein. Adipophilin occurs in a wide range of cultured cell lines, including fibroblasts and endothelial and epithelial cells. In tissues, however, expression of adipophilin is restricted to certain cell types, such as lactating mammary epithelial cells, adrenal cortex cells, Sertoli and Leydig cells of the male reproductive system, and steatosis or fatty change hepatocytes in alcoholic liver cirrhosis. Our results reveal adipophilin as a possible new marker for the identification of specialized differentiated cells containing lipid droplets and for diseases associated with fat-accumulating cells.

Adipocytes↗

PubMed1998

Molecular characterization of proteolytic processing of the Pol proteins of human foamy virus reveals novel features of the viral protease.

Spumaviruses, or foamy viruses, express a pol-specific transcript that codes for a Pol polyprotein that consists of the protease, reverse transcriptase, ribonuclease H, and the integrase domains. To delineate the proteolytic cleavage sites between the Pol subdomains, recombinant human foamy virus (HFV) Pol proteins were expressed, purified by affinity chromatography, and subjected to either HFV protease assays or autocatalytic processing. In control experiments, HFV protease-deficient mutant proteins in which the active site Asp was replaced by an Ala residue were used to rule out unspecific processing by nonviral proteases. Specific proteolytic cleavage products were isolated, and the cleavage sites were analyzed by amino acid sequencing. Peptides spanning the resulting cleavage sites were chemically synthesized and assayed with HFV protease, and the cleaved peptides were subjected to mass spectrometry. The cleavage site sequences obtained were in complete agreement with the amino-terminal sequences from amino acid sequencing of authentic cleavage products of the HFV Pol proteins. Analysis by fast-protein liquid chromatography of a short version of the active HFV protease revealed that the enzyme predominantly formed dimeric molecules.

Aspartic Acid Endopeptidases↗

PubMed1997

Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase.

In this paper we describe isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase (DAP-AT). The enzyme was extracted from rabbit Harderian gland peroxisomes and isolated as a trimeric complex by sucrose density gradient centrifugation. From peptide sequences matching EST-clones were obtained which allowed cloning and sequencing of the cDNA from a human cDNA library. The nucleotide-derived amino acid sequence revealed a protein consisting of 680 amino acid residues of molecular mass 77187 containing a C-terminal type 1 peroxisomal targeting signal. Monospecific antibodies raised against this polypeptide efficiently immunoprecipitated DAP-AT activity from solubilized peroxisomal preparations, thus demonstrating that the cloned cDNA codes for DAP-AT.

Acyltransferases↗

PubMed1997

Mediators of nuclear protein import target karyophilic proteins to pore complexes of cytoplasmic annulate lamellae.

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes of Xenopus laevis. In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin beta and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central "transporter" and closely resemble those of "transport intermediates" found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.

Animals↗

PubMed1997

CP beta3, a novel isoform of an actin-binding protein, is a component of the cytoskeletal calyx of the mammalian sperm head.

In the mammalian sperm head, the nucleus is tightly associated with the calyx, a cell type-specific cytoskeletal structure. Previously, we have identified and characterized some basic proteins such as calicin and cylicins I and II as major calyx components of bovine and human spermatids and spermatozoa. Surprisingly we have now discovered another calyx constituent which by amino acid sequencing and cDNA cloning was recognized as a novel isoform of the widespread beta subunit of the heterodimeric actin-binding "capping protein" (CP). This polypeptide, CP beta3, of sperm calices, is identical with the beta2 subunit present in diverse somatic cell types, except that it shows an amino-terminal extension of 29 amino acids and its mRNA is detected only in testis and, albeit in trace amounts, brain. This CP beta3 mRNA contains the additional sequence, encoded by exon 1 of the gene, which is missing in beta2 mRNAs. Antibodies specific for the beta3 amino-terminal addition have been used to identify the protein by immunoblotting and to localize it to the calyx structure by immunofluorescence microscopy. We conclude that in spermiogenesis the transcription of the gene encoding the beta1, beta2, and beta3 CP subunits is regulated specifically to include exon 1 and to give rise to the testis isoform CP beta3, which is integrated into the calyx structure of the forming sperm head. This surprising finding of an actin-binding protein isoform in an insoluble cytoskeletal structure is discussed in relation to the demonstrated roles of actin and certain actin-binding proteins, such as Limulus alpha-scruin, in spermiogenesis and spermatozoa.

Actin Depolymerizing Factors↗

PubMed1997

Identification of protein p270/Tpr as a constitutive component of the nuclear pore complex-attached intranuclear filaments.

Using a monoclonal antibody, mAb 203-37, we have identified a polypeptide of M(r) approximately 270 kD (p270) as a general constituent of the intranuclear filaments attached to the nucleoplasmic annulus of the nuclear pore complex (NPC) in diverse kinds of vertebrate cells. Using cDNA cloning and immunobiochemistry, we show that human protein p270 has a predicted molecular mass of 267 kD and is essentially identical to the coiled-coil dominated protein Tpr reported by others to be located on the outer, i.e., cytoplasmic surface of NPCs (Byrd, D.A., D.J. Sweet, N. Pante, K.N. Konstantinov, T. Guan, A.C.S. Saphire, P.J. Mitchell, C.S. Cooper, U. Aebi, and L. Gerace. 1994. J. Cell Biol. 127: 1515-1526). To clarify this controversial localization, we have performed immunoelectron microscopy in diverse kinds of mammalian and amphibian cells with a series of antibodies raised against different epitopes of human and Xenopus laevis p270/Tpr. In these experiments, the protein has been consistently and exclusively detected in the NPC-attached intranuclear filaments, and p270/Tpr-containing filament bundles have been traced into the nuclear interior for up to 350 nm. No reaction has been noted at the cytoplasmic side of NPCs with any of the p270/Tpr antibodies, whereas control antibodies such as those against protein RanBP2/Nup358 specifically decorate the cytoplasmic annulus of NPCs. Pore complexes of cytoplasmic annulate lamellae in various mammalian and amphibian cells are also devoid of immunodetectable protein p270/Tpr. We conclude that this coiled-coil protein is a general and ubiquitous component of the intranuclear NPC-attached filaments and discuss its possible functions.

3T3 Cells↗

PubMed1996

Comparative properties of feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV-1) proteinases prepared by total chemical synthesis.

The aspartyl proteinase (PR) encoded by the feline immunodeficiency virus (FIV) was prepared by total chemical synthesis. The 116-amino-acid polypeptide chain was assembled in a stepwise fashion using a Boc chemistry solid-phase peptide synthesis approach and subsequently folded into the biologically active dimeric proteinase. The synthetic enzyme showed proteolytic activity against a variety of different peptide substrates corresponding to putative cleavage sites of the Gag and Gag-Pol polyproteins of FIV. A comparative study with the proteinase of human immunodeficiency virus type 1 (HIV-1) showed that the FIV and HIV-1 enzymes have related but distinct substrate specificities. In particular, HIV-1 PR and FIV PR each show a strong preference for their own MA/CA substrates, despite identical amino acid residues at four of seven positions from P3-P4' of the substrate including an identical MA/CA cleavage site (between Tyr approximately Pro residues). FIV PR also showed a requirement for a longer peptide substrate than HIV-1 PR. Defining the similarities and the differences in the properties of these two retroviral enzymes will have a significant impact on structure-based drug design.

Amino Acid Sequence↗

PubMed1996

Specific immunohistochemical detection of cardiac/fetal alpha-actin in human cardiomyocytes and regenerating skeletal muscle cells.

We describe three murine monoclonal antibodies (mAbs) raised against a synthetic decapeptide representing the aminoterminal sequence of the cardiac/ fetal isoform of sarcomeric alpha-actin. When used for immunoblotting or histological immunolocalization, these mAbs distinguish cardiac/fetal alpha-actin from skeletal muscle alpha-actin, and also from all other actin isoforms. We show, by immunofluorescence and immunoperoxidase microscopy of tissue sections, that cardiac/fetal alpha-actin can be localized not only in cardiomyocytes but also in skeletal muscles and their satellite cells during regeneration. These mAbs are potentially valuable in developmental biology, for the characterization of tissue and cultured myogenic cells, in pathology, and for serodiagnosis.

Actins↗

PubMed1996

Patterns of desmocollin synthesis in human epithelia: immunolocalization of desmocollins 1 and 3 in special epithelia and in cultured cells.

Murine monoclonal antibodies (mAbs) which specifically react, in immunoblot and immunolocalization experiments, with the human desmosomal cadherins, desmocollins Dsc1 (mAb Dsc1-U100) and Dsc3 (mAb Dsc3-U114), have allowed to study systematically the synthesis of these proteins in tissues and cultured cells. Application of these mAbs in immunofluorescence microscopy on human skin has shown the presence of Dsc1 in the suprabasal layers of interfollicular epidermis and a specific cell layer of the hair follicle root sheath, whereas Dsc3 has been identified in all living epidermal layers as well as in glandular ducts and in basal matrix cells and the outer root sheath of hair follicles. Dsc3, but not Dsc1, it also present in desmosomes of the basal as well as suprabasal cell layers of other stratified epithelia such as vagina, tongue and esophagus as well as in cells of the basal layer of bladder urothelium and the complex epithelium of trachea. All the diverse one-layered ("simple") epithelia examined were as negative for both, Dsc1 and Dsc3, as were the non-epithelial desmosomes of the intercalated disks of the myocardium. A special situation has been discovered in the thymus. Here the usually single-layered cells of the thymic reticular epithelium are connected by Dsc3-possessing desmosomes, as they also contain typical (type I) hemidesmosomes, whereas Dsc1 is only detected in the "Hassall bodies", spheroidal formations of densely packed reticulum-derived cells which also produce cytokeratins 1 and 10, indicative of suprabasal epidermal differentiation. In cell cultures most, probably all, desmosomes of diverse cell lines derived from stratified squamous epithelia or squamous cell carcinomas, including primary keratinocytes, HaCaT keratinocytes and A-431 carcinoma cells, contain Dsc3. By contrast, Dsc1 has only been detected in local piles of keratinocytes that appear to be in the process of suprabasal differentiation. The antibodies have also allowed to demonstrate that desmosomes of cell lines can contain more than one desmocollin isoform. The observations made by immunofluorescence microscopy are compared with results obtained by in situ hybridization of mRNAs, and the potential value of these mAbs in histology and pathology is discussed.

Amino Acid Sequence↗

PubMed1985

Analysis of cosmids using linearization by phage lambda terminase.

A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments.

Animals↗

PubMed1984

Rapid restriction mapping of DNA cloned in lambda phage vectors.

A protocol for the rapid restriction mapping of phage lambda clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive lambda DNA termini by hybridisation with [32P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the "ladder" of partial digestion products.

Bacteriophage lambda↗

PubMed1984

Selective isolation of cosmid clones by homologous recombination in Escherichia coli.

A procedure for selection of specific cosmid clones by homologous recombination between cosmid clones from a library and sequences cloned into a plasmid has been developed. Cosmid libraries constructed in a rec- host strain are packaged in vivo into lambda particles. Appropriate aliquots are then introduced into a rec+ host containing the sequence used for selection cloned into a plasmid vector without sequence homology to the cosmid vector. After a short time for recombination, the cosmids are packaged in vivo. Cosmids that have taken up the plasmid by homologous recombination are isolated by plating under conditions selecting for the antibiotic resistance markers carried by both vectors. The recombined cosmids can lose the inserted sequence by another homologous recombination event and, after packaging in vivo, these revertants can be identified on appropriate indicator plates.

Animals↗