Biomedical subjects

C Kannicht

Publications and source records attributed to C Kannicht.

12 recordsLinked to original sources

PubMed2006

Characterisation of a novel high-purity, double virus inactivated von Willebrand Factor and Factor VIII concentrate (Wilate).

This study summarises the biochemical and functional properties of a new generation plasma-derived, double virus inactivated von Willebrand Factor/Factor VIII (VWF/FVIII) concentrate, Wilate, targeted for the treatment of both von Willebrand disease (VWD) and haemophilia A. The manufacturing process comprises two chromatographic steps based on different performance principles, ensuring a high purity of the concentrate (mean specific activity in 15 consecutive production batches: 122 IU FVIII:C/mg total protein) and, thus, minimising the administered protein load to the patient (specification: < or = 15 mg total protein per 900 IU Wilate). The optimised solvent/detergent (S/D) treatment and prolonged terminal dry-heat (PermaHeat) treatment of the lyophilised product at a specified residual moisture (RM) provide two mechanistically independent, effective and robust virus inactivation procedures for enveloped viruses and one step for non-enveloped viruses. These process steps are aggressive enough to inactivate viruses efficiently, but yet gentle enough to maintain the structural integrity and function of the VWF and FVIII molecules, as proven by state-of-the-art assays covering the diverse features of importance. The VWF multimeric pattern is close to the one displayed by normal plasma, with a consistent content of more than 10 multimers, but a relatively lower portion of the very high multimers. The multimeric triplet structure is normal, underlining the gentle and effective manufacturing process, which does not require the addition of protein stabilisers at any step. The balanced activity ratio of VWF to FVIII is close to that of plasma from healthy subjects, rendering Wilate suitable also for the safe and effective treatment of patients with VWD.

Blood Proteins↗

PubMed2001

Vitronectin in clotting factor IX concentrates.

Highly purified, plasma-derived factor IX (FIX) concentrates are produced in large part by a combination of anion exchange and heparin affinity chromatography. However, the concentrates still contain some accompanying proteins. The main impurity has turned out to be the adhesive glycoprotein, vitronectin. It occurs in concentrates exclusively in its multimeric form, in contrast to the situation in plasma. The multimeric vitronectin can be removed either by nanofiltration with a crossflow system or by size-exclusion chromatography. When these FIX concentrates are used as therapeutic agents, the fact has to be taken into account that considerable amounts of multimeric vitronectin are given to the patient. The physiological consequences of the dosage of this protein have not yet been investigated. Although no thrombogenicity has been reported in connection with the above-mentioned FIX concentrates, we recommend that the impurity should be removed from the preparation with the methods described here.

Blood Coagulation Factors↗

PubMed1999

N-glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures.

Rat C-CAM is a ubiquitous, transmembrane and carcinoembryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glycosylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be oversialylated.

Adenosine Triphosphatases↗

PubMed1999

Degradation products of factor VIII which can lead to increased immunogenicity.

The biochemical and immunochemical aspects of the development of inhibitors with a plasma-derived, double-virus inactivated factor VIII (FVIII) concentrate (marketed as Octavi SDPlus in Germany and Bisinact in Belgium) are described. A total of 12 cases of inhibitor formation (predominantly type II) were reported in Germany, 8 in Belgium but none in Portugal. Initially, the only difference between the non-pasteurised, SD virus-inactivated product Octavi and the pasteurised product Octavi SDPlus appeared to be pasteurisation, though subsequently, the quality of source material for the product was found to differ in different countries. Separation studies revealed the presence of a 40 kDa peptide fragment in some batches. It was subsequently shown that there was a strong correlation between inhibitor development and batches containing the 40 kDa marker, and a relationship between elevated markers of coagulation activation (FPA in particular) and the occurrence of the 40 kDa marker. Further work revealed that analytical methods commonly used for quality control were not suitable to highlight batch-to-batch differences. It was concluded that inhibitor potential (neoantigenicity) in Octavi SDPlus arose due to two effects; degradation of FVIII already present in source material; and heating of unstable FVIII degradation products. In this case, inhibitors were not caused by the overall production process, nor by GMP failures. The problem of inhibitor potential can be avoided if appropriate preventive measures are taken. Further work is needed to prove non-neoantigenicity and to reinforce the scientific findings, and to characterise pilot batches.

Chromatography, High Pressure Liquid↗

PubMed1998

Cell surface glycoproteins undergo postbiosynthetic modification of their N-glycans by stepwise demannosylation.

Primary rat hepatocytes and two hepatoma cell lines have been used to study whether high mannose-type N-glycans of plasma membrane glycoproteins may be modified by the removal of mannose residues even after transport to the cell surface. To examine glycan remodeling of cell surface glycoproteins, high mannose-type glycoforms were generated by adding the reversible mannosidase I inhibitor deoxymannojirimycin during metabolic labeling with [3H]mannose, thereby preventing further processing of high mannose-type N-glycans to complex structures. Upon transport to the cell surface, glycoproteins were additionally labeled with sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate. This strategy allowed us to follow selectively the fate of cell surface glycoproteins. Postbiosynthetic demannosylation was monitored by determining the conversion of Man8-9GlcNAc2 to smaller structures during reculture of cells in the absence of deoxymannojirimycin. The results show that high mannose-type N-glycans of selected cell surface glycoproteins are trimmed from Man8-9GlcNAc2 to Man5GlcNAc2 with Man7GlcNAc2 and Man6GlcNAc2 formed as intermediates. It could be clearly shown in MH 7777 as well as in HepG2 cells that demannosylation affects plasma membrane glycoproteins after they are routed to the cell surface. As was determined for total cell surface glycoproteins in HepG2 cells, this process occurs with a half-time of 6.7 h. By analyzing the size of high mannose-type glycans of glycoproteins isolated from the cell surface at the end of the reculture period, i.e. after trimming had occurred, we were able to demonstrate that glycoproteins carrying trimmed high mannose glycans become exposed at the cell surface. From these data we conclude that cell surface glycoproteins can be trimmed by mannosidases at sites peripheral to N-acetylglucosaminyltransferase I without further processing of their glycans to the complex form. This glycan remodeling may occur at the cell surface or during endocytosis and recycling back to the cell surface.

Animals↗

PubMed1997

Analysis of site-specific N-glycosylation of recombinant Desmodus rotundus salivary plasminogen activator rDSPA alpha 1 expressed in Chinese hamster ovary cells.

The recombinant plasminogen activator (rDSPA alpha 1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhibits a superior pharmacological profile if compared to tissue-type plasminogen activator (t-PA) or streptokinase. In the present study the structures of the carbohydrate moieties at the two N-glycosylation sites (Asn-117, Asn-362) of rDSPA alpha 1 expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F digestion and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by sequence analysis in which the fluorescently labeled glycans were cleaved with an array of specific exoglycosidases. More than 30 different oligosaccharides were identified. The results revealed that Asn-117 carried a mixture of one high-mannose structure (17% of site-specific glycosylation), three hybrid glycans (26%) and predominantly biantennary complex N-glycans (54%). Glycosylation site Asn-362 was found to comprise complex glycans with biantennary (50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennary structures (10%), which were fucosylated at the innermost residue of N-acetylglucosamine. Mainly neutral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites. Sialic acid was alpha 2-3 linked to galactose exclusively. As shown in this study the N-glycans attached to Asn-117 of rDSPA alpha 1 are more processed during biosynthesis than the high-mannose structures linked to Asn-117 of t-PA, to which the polypeptide backbone of rDSPA alpha 1 is structurally closely related.

Amino Acid Sequence↗

PubMed1996

O-linked L-fucose is present in Desmodus rotundus salivary plasminogen activator.

DSPAalpha1 (Desmodus rotundus salivary plasminogen activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An unusual type of posttranslational modification, in which L-fucose is O-glycosidically linked to threonine 61 in the epidermal growth factor domain was found for natural DSPAalpha1 and its recombinant form isolated from Chinese hamster ovary cells. In the present study a combination of carbohydrate and amino acid composition analysis, amino acid sequencing, and mass spectrometry revealed that the L-fucose is bound to residues 56-68 of DSPAalpha1. The amino acid sequence of this glycosylation site agreed with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys described for other proteins. Anew strategy for the identification of the modified amino acid was established. Direct evidence for the occurrence of fucosyl-threonine was obtained by mass spectrometry after digestion of the glycopeptide with a mixture of peptidases. On the basis of these results, DSPAalpha1 is a suitable model for studying the influence of O-fucosylation on clearance rates, particularly in comparative studies with the identically fucosylated and structurally related tissue plasminogen activator.

Amino Acid Sequence↗

PubMed1993

Synthesis of 2-deoxy-D-galactose containing gangliosides in vivo.

Incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of different gangliosides of rat liver was examined. After intraperitoneal administration of 2-deoxy-D-galactose it was shown by GLC/MS analysis that this hexose analogue is metabolized and incorporated into all the gangliosides investigated, and predominantly into GM3 and GD3. In both of these gangliosides, 25-55% of the galactose residues were substituted by 2-deoxy-D-galactose. The epimer, 2-deoxy-D-glucose, was not detectable.

Animals↗

PubMed1993

Characterisation of the ATP-dependent taurocholate-carrier protein (gp110) of the hepatocyte canalicular membrane.

The canalicular domain-specific glycoprotein gp110, which recently has been shown to function as an ATP-dependent taurocholate transporter, has been purified 1800-fold from rat liver plasma membranes. gp110 has been characterised as an integral plasma membrane protein with M(r) of 100,000-115,000 and pI of 2.5-3.5 and possesses a highly glycosylated and negatively charged extra-cellular domain. The broad range of M(r) and pI values results from the existence of numerous glycoforms composed of sialylated N-glycans. After deglycosylation, the polypeptide has M(r) 48,000 and pI 5.0. In primary cultures of rat hepatocytes, gp110 is synthesised with M(r) 110,000, while in the presence of tunicamycin the non-glycosylated form has M(r) 48,000. In the presence of 1-deoxymannojirimycin, two forms of M(r) 83,000 and M(r) 91,000 were found, which were converted by endo-beta-N-acetylglucosaminidase H into a single 52,000-M(r) band, indicating the existence of two basic glycoforms at the oligomannosyl stage of biosynthesis. gp110 was phosphorylated at serine residues in primary cultures of hepatocytes. The sequences of ten internal peptides of gp110 were identical to the sequence of the high-M(r) form of ecto-ATPase, but ecto-ATPase activity from plasma-membrane extracts was not depleted by anti-(gp110) serum. In contrast, Fab fragments of these antibodies inhibit the aggregation of freshly isolated hepatocytes.

ATP-Binding Cassette Transporters↗

PubMed1992

Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors.

In this study it could be shown that in rat the normally occurring N-acetyl neuraminic acid can be modified in its N-acyl moiety by in vivo administration of the chemically synthesized N-propanoyl precursors, N-propanoyl-D-glucosamine or N-propanoyl-D-mannosamine. It could be shown that each of these nonphysiological amino sugar analogues was incorporated into both membrane and serum glycoproteins. After treatment of rats with radiolabeled N-[acyl-1-14C]D-mannosamine, radioactivity could be removed from serum glycoprotein fractions by incubation with neuraminidase from Clostridium perfringens or from Arthrobacter ureafaciens. Mild acid hydrolysis removed 98% of the radioactivity after in vivo labeling with N-[acetyl-1-14C]D-mannosamine and 86% after labeling with N-[propanoyl-1-14C]D-mannosamine. Chromatographic analysis yielded two compounds, i.e. N-acetyl neuraminic acid and N-propanoyl neuraminic acid, the latter being identified by gas liquid chromatography/mass spectrometry studies. Measurement of protein-bound radioactivity in different rat organs revealed a different organotropy of the natural and the nonphysiological neuraminic acid precursor. Of the glucosamine derivatives, N-acetyl-D-glucosamine showed the higher rate of uptake and incorporation in most organs (except in the submandibulary gland), and especially in kidney cortex and Morris hepatoma 7777. Natural and the unphysiological mannosamine derivatives were incorporated at similar rates, except in liver, where N-acetyl-D-mannosamine was taken up and metabolized more effectively. This finding indicates that it is possible to modify the acyl group of N-acetyl neuraminic acid in vivo by the introduction of an N-propanoyl group and possibly other homologous N-acyl groups. This procedure may provide a tool for a further characterization of the biological function of sialic acids.

Animals↗

PubMed1992

Incorporation of the hexose analogue 2-deoxy-D-galactose into membrane glycoproteins in HepG2 cells.

The incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of glycoproteins and the consequences of 2-deoxy-D-galactose treatment on the fucosylation of glycoproteins were investigated in the human hepatoma cell line HepG2. Using different methods, it was shown that treatment of HepG2 cells with 2-deoxy-D-galactose leads to an incorporation of 2-deoxy-D-galactose and a decrease of L-fucose incorporation into the oligosaccharides of glycoproteins. The extent of labeling by L-[3H]fucose was determined by removing L-[3H]fucose from labeled cells with the aid of a purified alpha 1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy-D-galactose markedly inhibits alpha 1,2-fucosylation. Measurement of the amount of 2-deoxy-D-galactose incorporated, however, showed that replacement of D-galactose by 2-deoxy-D-galactose does not entirely account for the decrease in alpha 1,2-fucosylation. In addition, a hitherto unreported compensatory increase of alpha 1,3/alpha 1,4-fucosylation was found to occur when alpha-1,2-fucosylation was inhibited by treatment with 2-deoxy-D-galactose.

Adenosine Triphosphate↗

PubMed1991

ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt.

Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems.

ATP-Binding Cassette Transporters↗