Abstrakts & Konferenz-Beiträge
|Biochim Biophys Acta 1988 Mar 11;968(3):331-9||Related Articles, Books|
Goldenberg H, Eder M, Pumm R, Wallner E, Retzek H, Huttinger M
Department of Medical Chemistry, University of Vienna, Austria.
Radioactively labelled transferrin was injected into rats intravenously and its uptake and subcellular distribution in the liver was investigated. The amount of radioactivity in the liver remained constant from 10 min after injection. It was not influenced by asialoglycoproteins. The radioactive label was identified as asialotransferrin. After subcellular fractionation by differential and zonal sucrose density-gradient centrifugation the label was enriched in a low-density endocytic compartment showing fluorescence quenching of acridine orange and N-ethylmaleimide-sensitive ATPase activity. The data fit into a model of continuous transferrin-receptor-mediated recycling through the hepatocyte’s endocytic compartment.
|DNA Cell Biol 1992 Nov;11(9):661-72||Related Articles, Books, Protein, Nucleotide|
Retzek H, Steyrer E, Sanders EJ, Nimpf J, Schneider WJ
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Upon receptor-mediated endocytosis of very-low-density lipoprotein (VLDL) and vitellogenin into growing chicken oocytes, the protein moieties of these lipoproteins are proteolytically cleaved. Unlike the complete lysosomal degradation in somatic cells, enzymatic ligand breakdown in oocytes generates a characteristic set of polypeptides, which enter yolk storage compartments for subsequent utilization by the embryo. Here, we demonstrate directly that the catalyst for the intraoocytic processing of both apolipoprotein B and vitellogenin is cathepsin D. The enzyme was purified from oocytic yolk, preovulatory follicle homogenates, and liver by affinity chromatography. When plasma VLDL and vitellogenin were incubated with the purified enzyme, fragments indistinguishable from those found in yolk were generated from both precursors under identical, mildly acidic conditions. Amino-terminal sequencing of the pure enzyme demonstrated 88% identity with mammalian cathepsin Ds over 34 residues. On the basis of this information, a full-length clone specifying chicken preprocathepsin D was isolated from a chicken follicle cDNA library by screening with a human cathepsin D probe. Whereas previous studies have demonstrated that the receptors for lipoproteins in somatic cells and oocytes, respectively, of the chicken are the products of different genes, Southern and Northern blot hybridization experiments showed that the enzymes expressed in oocytes and liver are the product of a single gene, giving rise to a 3.3-kb transcript. The primary structure of the 335-residue mature protein suggests a hig
h degree of conservation of known crucial features of aspartyl proteases; however, the absence of the so-called processing region and of an aromatic residue in a region thought to partake in catalysis raise questions with possible evolutionary implications.
|Clin Biochem 1988 Apr;21(2):87-92||Related Articles, Books|
Huettinger M, Retzek H, Eder M, Goldenberg H
University of Vienna, Department of Medical Chemistry, Austria.
We have investigated uptake of 125I-labeled chylomicron remnants into livers of rats in the presence of lactoferrin. This glycoprotein possesses a cluster of four arginines at the N-terminus similar to the arginine rich binding sequence of apoprotein E (apoE) to the LDL-receptor. We found that this protein inhibits uptake of 125I-chylomicron remnant radioactivity by 50% when measured as accumulation of radioactivity into the intact organ, and even more pronounced, over 75%, when measured as uptake into an endosomal fraction prepared therefrom. Provided that the arginine rich sequence is responsible for the inhibition, a similarity in the characteristics of binding of apoE to the LDL (low density lipoprotein)- and chylomicron remnant-receptor is likely. Second, transferrin having sequence homologies with lactoferrin, but lacking the arginine cluster does not interfere with chylomicron remnant uptake. Third, lactoferrin does not inhibit the uptake of chylomicron remnants by the spleen, which is most likely mediated through scavenger cells by a mechanism different from the chylomicron remnant uptake system of the liver. We hypothesize from this that lactoferrin specifically interferes with the physiologically relevant chylomicron remnant uptake system of the liver. Investigation of the mechanism of this inhibition will provide information about the physical characteristics of the remnant receptor system.
|: Cell Tissue Res 1993 Jun;272(3):459-71||Related Articles, Books, LinkOut|
Shen X, Steyrer E, Retzek H, Sanders EJ, Schneider WJ
Department of Medicine, University of Manitoba, Winnipeg, Canada.
During the rapid final stage of growth, chicken oocytes take up massive amounts of plasma components and convert them to yolk. The oocyte expresses a receptor that binds both major yolk lipoprotein precursors, vitellogenin (VTG) and very low density lipoprotein (VLDL). In the present study, in vivo transport tracing methodology, isolation of coated vesicles, ligand- and immuno-blotting, and ultrastructural immunocytochemistry were used for the analysis of receptor-mediated yolk formation. The VTG/VLDL receptor was identified in coated profiles in the oocyte periphery, in isolated coated vesicles, and within vesicular compartments both outside and inside membrane-bounded yolk storage organelles (yolk spheres). VLDL particles colocalized with the receptor, as demonstrated by ultrastructural visualization of VLDL-gold following intravenous administration, as well as by immunocytochemical analysis with antibodies to VLDL. Lipoprotein particles were shown to reach the oocyte surface by passage across the basement membrane, which possibly plays an active and selective role in yolk precursor accessibility to the oocyte surface, and through gaps between the follicular granulosa cells. Following delivery of ligands from the plasma membrane into yolk spheres, proteolytic processing of VTG and VLDL by cathepsin D appears to correlate with segregation of receptors and ligands which enter disparate sub-compartments within the yolk spheres. In small, quiescent oocytes, the VTG/VLDL receptor was localized to the central portion of the cell. At onset of the rapid growth phase, it appears that this pre-existing pool of receptors redistributes to the peripheral region, thereby initiating yolk formation. Such a redistribution mechanism would obliterate the need for de novo synthesis of receptors when the oocyte’s energy expenditure is to be utilized for plasma membrane synthesis, establishment and maintenance of intracellular topography and yolk formation, and preparation for ovulation.
|J Biol Chem 1992 Sep 15;267(26):18551-7||Related Articles, Books|
Huettinger M, Retzek H, Hermann M, Goldenberg H
Department of Medical Chemistry, University of Vienna, Austria.
Our recently found nonlipoprotein inhibitor of chylomicron remnant uptake, lactoferrin, has been investigated in vivo and in vitro. Lipoprotein lipase extracted triglycerides from chylomicrons, doubly labeled with [3H]retinol/[14C]oleate, in the presence of lactoferrin normally. The subsequent uptake of remnants into liver was retarded considerably. In the intact rat, chylomicron remnants (CRs), predominantly labeled in the apoB48 moiety by 125I, were excluded from the hepatic endosomal compartment in the presence of lactoferrin as shown in subcellular fractionation studies of rat livers. In tissue culture, internalization of [125I]chylomicron remnants was inhibited in the presence of 14 pM lactoferrin by 70%. Upon removal of lactoferrin, internalization was rapidly restored. Protease digestion eliminated the inhibitory effect completely. Modification of arginine residues with cyclohexanedione reversibly removed the inhibitory potency of lactoferrin. We located by molecular modeling an alpha-helical segment in lactoferrin on the exposed surface of the molecule containing the sequence Arg-X-X-Arg-Lys-X-Arg, which resembles the receptor recognition structure in apolipoprotein E (apoE). This firmly established ligand correspondence with apoE, the candidate ligand for CR recognition by the receptor. Finally, the postulated second function of low density lipoprotein receptor-related protein, uptake of alpha-2-macroglobulin (alpha 2M) was found to be distinct from lipoprotein binding, since lactoferrin inhibited CR but not alpha 2M internalization.
In addition, CR uptake was not affected by alpha 2M. We conclude that if a bifunctional receptor were to operate, its diverse functions were exerted by independently operating substructures. The results of our in vivo and cell culture experiments are, however, entirely compatible with the existence of two receptors as well.
Cloning Result of Chicken Cathepsin D
NiceProt – a user-friendly view of this SWISS-PROT entry
ID CATD_CHICK STANDARD; PRT; 398 AA.
DT 01-FEB-1994 (REL. 28, CREATED)
DT 01-FEB-1994 (REL. 28, LAST SEQUENCE UPDATE)
DT 01-NOV-1995 (REL. 32, LAST ANNOTATION UPDATE)
DE CATHEPSIN D PRECURSOR (EC 22.214.171.124).
OS GALLUS GALLUS (CHICKEN).
OC EUKARYOTA; METAZOA; CHORDATA; VERTEBRATA; ARCHOSAURIA; AVES;
OC NEOGNATHAE; GALLIFORMES; PHASIANIDAE; PHASIANINAE; GALLUS.
RP SEQUENCE FROM N.A., AND SEQUENCE OF 64-97.
RX MEDLINE; 93039672. [NCBI, ExPASy, Israel, Japan]
RA RETZEK H., STEYRER E., SANDERS E.J., NIMPF J., SCHNEIDER W.J.;
RT „Molecular cloning and functional characterization of chicken
RT cathepsin D, a key enzyme for yolk formation.“;
RL DNA CELL BIOL. 11:661-672(1992).
CC -!- FUNCTION: CATHEPSIN D IS AN ACID PROTEASE ACTIVE IN INTRACELLULAR
CC PROTEIN BREAKDOWN. IN CHICKEN IT IS A KEY ENZYME FOR YOLK
CC FORMATION AS IT IS CAPABLE OF CATALYZING INTRA OOCYTIC BREAK DOWN
CC OF PROTEIN COMPONENTS OF BOTH VITELLOGENIN AND VLDL.
CC -!- SUBUNIT: CONSISTS OF A LIGHT CHAIN AND A HEAVY CHAIN.
CC -!- SUBCELLULAR LOCATION: LYSOSOMAL.
CC -!- TISSUE SPECIFICITY: OOCYTIC YOLK, PREOVULATORY FOLLICLES, LIVER.
CC -!- SIMILARITY: BELONGS TO PEPTIDASE FAMILY A1; ALSO KNOWN AS THE
CC EUKARYOTIC ASPARTYL PROTEASES FAMILY.
CC This SWISS-PROT entry is copyright. It is produced through a collaboration
CC between the Swiss Institute of Bioinformatics and the EMBL outstation –
CC the European Bioinformatics Institute. There are no restrictions on its
CC use by non-profit institutions as long as its content is in no way
CC modified and this statement is not removed. Usage by and for commercial
CC entities requires a license agreement (See http://www.isb-sib.ch/announce/
CC or send an email to email@example.com).
DR EMBL; S49650; AAB24157.1; -. [EMBL / GenBank / DDBJ] [CoDingSequence]
DR PROSITE; PS00141; ASP_PROTEASE; 2.
DR PFAM; PF00026; asp; 1.
DR HSSP; P07339; 1LYA. [HSSP ENTRY / SWISS-3DIMAGE /
DR PDB-ENTRY / PDB-RASMOL / PDB-3DIMAGE]
DR DOMO; Q05744.
DR PRODOM [Domain structure / List of seq. sharing at least 1 domain]
DR PROTOMAP; Q05744.
DR PRESAGE; Q05744.
DR SWISS-2DPAGE; GET REGION ON 2D PAGE.
KW HYDROLASE; ASPARTYL PROTEASE; GLYCOPROTEIN; LYSOSOME; SIGNAL; ZYMOGEN.
FT SIGNAL 1 20 POTENTIAL.
FT PROPEP 21 63 ACTIVATION PEPTIDE (POTENTIAL).
FT CHAIN 64 398 CATHEPSIN D.
FT CHAIN 64 157 LIGHT CHAIN (PROBABLE).
FT CHAIN 158 398 HEAVY CHAIN (PROBABLE).
FT ACT_SITE 96 96 BY SIMILARITY.
FT ACT_SITE 283 283 BY SIMILARITY.
FT DISULFID 109 116 BY SIMILARITY.
FT DISULFID 274 278 BY SIMILARITY.
FT DISULFID 317 354 BY SIMILARITY.
FT CARBOHYD 133 133 POTENTIAL.
FT CARBOHYD 251 251 POTENTIAL.
SQ SEQUENCE 398 AA; 43298 MW; A45111DE CRC32;
MAPRGLLVLL LLALVGPCAA LIRIPLTKFT STRRMLTEVG SEIPDMNAIT QFLKFKLGFA
DLAEPTPEIL KNYMDAQYYG EIGIGTPPQK FTVVFDTGSS NLWVPSVHCH LLDIACLLHH
KYDASKSSTY VENGTEFAIH YGTGSLSGFL SQDTVTLGNL KIKNQIFGEA VKQPGITFIA
AKFDGILGMA FPRISVDKVT PFFDNVMQQK LIEKNIFSFY LNRDPTAQPG GELLLGGTDP
KYYSGDFSWV NVTRKAYWQV HMDSVDVANG LTLCKGGCEA IVDTGTSLIT GPTKEVKELQ
TAIGAKPLIK GQYVISCDKI SSLPVVTLML GGKPYQLTGE QYVFKVSAQG ETICLSGFSG
LDVPPPGGPL WILGDVFIGP YYTVFDRDND SVGFAKCV
NiceProt – a user-friendly view of this SWISS-PROT entry
Report form for errors/updates in this SWISS-PROT entry
Q05744 in FASTA format