Biomedical subjects

J Wölle

Publications and source records attributed to J Wölle.

10 recordsLinked to original sources

PubMed2001

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Animals↗

PubMed2000

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.

Amino Acid Sequence↗

PubMed1996

Transient overexpression of human 15-lipoxygenase in aortic endothelial cells enhances tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression.

15-lipoxygenase (15-LO) expression in artery wall cells has been demonstrated during the development of atherosclerosis in various animal models. We examined whether the expression of 15-LO in aortic endothelial cells affects the gene expression of the adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1). Transient transfection of human 15-LO cDNA into bovine aortic endothelial cells led to the expression of 15-LO protein and enzymatic activity. We studied the induction of VCAM-1 mRNA in these cells. 15-LO expressing cells showed no detectable levels of VCAM-1 message. However, when TNF was added to these cells there was a synergistic increase in VCAM-1 expression relative to cells that were transfected with control plasmid pcDNA I. Our data suggest that 15-LO expression in aortic endothelium may amplify the expression of VCAM-1 induced by inflammatory stimulus during atherogenesis.

Animals↗

PubMed1996

Oxidation of low density lipoproteins greatly enhances their association with lipoprotein lipase anchored to endothelial cell matrix.

Native and oxidized low density lipoprotein retention within arterial wall endothelial cell matrix (ECM) is an early event in the pathogenesis of atherosclerosis. Previously we showed lipoprotein lipase (LPL) addition to ECM enhanced the retention of apoB-containing lipoproteins. In the present studies we examined whether the oxidation of low density lipoprotein (LDL) increases its retention by LPL-containing ECM. Except where noted, 125I-labeled moderately oxidized LDL (ModOxLDL) was prepared by long term storage of 125I-LDL. Without LPL, 125I-ModOxLDL matrix binding was low and nonsaturable. LPL preanchored to ECM resulted in 125I-ModOxLDL binding that was saturable and 20-fold greater than in the absence of LPL, with an association constant equal to 2.6 nM. Copper-oxidized LDL (Cu-OxLDL) was able to compete with 125I-ModOxLDL, whereas a 60-fold native LDL excess had no effect. Reconstituted apolipoprotein B from Cu-OxLDL also reduced 125I-ModOxLDL to LPL, whereas liposomes derived from the lipid extract of Cu-OxLDL had no effect on binding. These data suggest that the increased binding of oxidized LDL to LPL-ECM may be due to the exposure of novel apoB binding sites and not an oxidized lipid moiety. 125I-ModOxLDL binding was also not affected by either preincubation with a 300-fold molar excess of apoE-poor HDL or an 340-fold molar excess of Cu-Ox-HDL. In contrast, a 4-fold apoE-rich HDL excess (based on protein) totally inhibited 125I-ModOxLDL matrix retention. Positively charged peptides of polyarginine mimicked the effect of apoE-rich HDL in reducing the 125I-ModOxLDL retention; however, polylysine had no effect. We postulate that the oxidation of LDL may be a mechanism that enhances LDL retention by the ECM-bound LPL and that the protective effects of apoE-containing HDL may in part be due to its ability to block the retention of oxidized LDL in vivo.

Animals↗

PubMed1996

Selective inhibition of tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid. Lack of effect on transcription factor NF-kappa B.

In the present studies, we examined the effect of flavonoids on the endothelial cell expression of adhesion molecules, an early step in inflammation and atherogenesis. Addition of tumor necrosis factor-alpha (TNF) to human aortic endothelial cells (HAECs) led to the induction of vascular cell adhesion molecule-1 (VCAM-1) expression and enhancement in expression of intercellular adhesion molecule-1 (ICAM-1). A flavonoid, 2-(3-amino-phenyl)-8-methoxy-chromene-4-one (PD 098063), markedly inhibited TNF-induced VCAM-1 cell-surface expression in a concentration-dependent fashion with half-maximal inhibition at 19 mumol/L but had no effect on ICAM-1 expression. Another structurally distinct flavonoid, 2-phenyl-chromene-4-one, similarly selectively decreased VCAM-1 expression. The inhibition in cell-surface expression of VCAM-1 by PD 098063 correlated with decreases in steady-state mRNA levels, but there was no effect on ICAM-1 mRNA levels. The decrease in VCAM-1 mRNA levels was not due to changes in mRNA stability but rather resulted from a reduction in the rate of transcription of the gene. However, electrophoretic mobility shift assays using nuclear extracts from TNF-induced HAECs treated with PD 098063 failed to show a decrease in the activation of NF-kappa B, indicating that inhibition of activation of this transcription factor may not be its mode of action. Similarly, PD 098063 did not affect chloramphenicol acetyltransferase reporter gene activity in TNF-inducible minimal VCAM-1 promoter constructs containing two NF-kappa B sites, suggesting that the compound does not affect the transactivation driven by these sites. We conclude that this compound selectively blocks agonist-induced VCAM-1 protein and gene expression in HAECs by NF-kappa B-independent mechanism(s).

Cells, Cultured↗

PubMed1995

Identification of a novel 85-kDa lipoprotein lipase binding protein on human aortic endothelial cell surface.

Lipoprotein lipase (LPL), bound to the luminal surface of vascular endothelium catalyzes lipoprotein triglyceride hydrolysis. Studies were performed to identify human aortic endothelial (HAEC) cell-surface proteins having high affinity for LPL. LPL-sepharose affinity chromatography of [35S]O4 labeled HAEC proteins identified a 220-kDa proteoglycan. Ligand blotting of HAEC plasma membrane proteins with LPL revealed two specific binding proteins of MW 116 kDa and 85 kDa, respectively, which were not released from the cell-surface by heparin treatment. Since the 220-kDa and 116-kDa proteins have been reported previously in bovine endothelial cells, we focused on the 85-kDa protein. The 85-kDa protein was not labelled by incubation of the cells with [35S]O4, suggesting that it is not a sulfated proteoglycan. Treatment of HAEC with tunicamycin markedly decreased detection of the 85-kDa protein, suggesting that it is likely a glycoprotein synthesized by HAEC. We conclude that HAEC cell surface has three specific LPL binding proteins, a 220-kDa proteoglycan, a 116-kDa protein and a novel 85-kDa protein.

Aorta↗

PubMed1995

Inhibition of tumor necrosis factor induced human aortic endothelial cell adhesion molecule gene expression by an alkoxybenzo[b]thiophene-2-carboxamide.

The effects of a novel anti-inflammatory agent, 5-methoxy-3-(1-methyl-ethoxy)benzo[b]thiophene-2-carboxamide-1-oxide (PD 144795) on adhesion molecule expression in tumor necrosis factor (TNF) stimulated human aortic endothelial cells (HAEC) were examined. PD 144795 treatment markedly inhibited the TNF-induced cell expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein and mRNA. Gel shift assays using nuclear extracts from HAEC treated with PD 144795 failed to show a decrease in the activation of NFkB by this compound, whereas pyrrolidine dithiocarbamate (PDTC), an antioxidant, markedly inhibited the activation of this transcription factor. Thus, PD 144795 inhibits agonist-stimulated VCAM-1 and ICAM-1 expression likely via an NFkB independent mechanism, distinct from that of PDTC. Such agents may provide a novel approach for control of adhesion molecule gene expression in inflammation.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1995

Apolipoprotein B and E basic amino acid clusters influence low-density lipoprotein association with lipoprotein lipase anchored to the subendothelial matrix.

Lipoprotein accumulation in the subendothelial matrix is an important step in atherogenesis. We have previously shown that addition of lipoprotein lipase (LPL) markedly increased binding of apolipoprotein B (apoB)-containing lipoproteins to an endothelial cell-derived matrix, and this enhanced lipoprotein binding was inhibited by apoE. In the present studies we examined the role of various regions of apoB in the binding of LDL to LPL-containing endothelial cell matrix and the ability of various apoE domains to decrease lipoprotein retention. We studied three apoB epitope-specific monoclonal antibodies for their ability to block the binding of 125I-LDL to LPL-containing matrix. Of these, monoclonal antibody 4G3, which recognizes an arginine-containing epitope in apoB, was the most effective in reducing LDL binding. Chemical modification of LDL apoB lysines or arginines markedly reduced the ability of the lipoprotein to block the binding of 125I-LDL to LPL-containing matrix, suggesting that apoB positively charged amino acids are involved in the interaction. Furthermore, polyarginine or polylysine markedly decreased 125I-LDL binding to LPL-containing matrix, whereas polyleucine was ineffective. These data suggest that apoB positively charged regions are important in LDL binding. To explore the role of charge modifications on apoE by single arginine-cysteine interchanges, we examined the effects of the three major human apoE isoforms (apoE2, apoE3, and apoE4). ApoE3 was the most effective in decreasing 125I-LDL retention, followed by apoE4; apoE2 was the least effective. Similarly, apoE2-containing HDL was much less effective than apoE3-containing HDL in decreasing 125I-LDL retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗

PubMed1993

Functional role of N-linked glycosylation in human hepatic lipase: asparagine-56 is important for both enzyme activity and secretion.

Hepatic lipase (HL) and lipoprotein lipase (LPL) are evolutionarily related enzymes that are essential for normal lipoprotein metabolism. While much has been published on the structure-function relationship of LPL, little is known concerning the structural basis of HL action and secretion. Human HL is a glycoprotein and its predicted amino acid sequence contains four putative N-linked glycosylation sites at Asn residues 20, 56, 340, and 375. We studied the role of these residues in the secretion and catalytic activity of hHL by analysis of hHL expressed in stable CHO cell lines. Using site-specific mutagenesis, the wild-type human HL and substitution mutants of each of the four Asn residues were expressed in vitro. The relative sizes of these site-specific mutants indicate that all four putative sites are utilized for glycosylation in CHO cells. Abolition of N-linked glycosylation of three (residues 20, 340, and 375) of the four sites did not affect enzyme secretion or activity. Mutations of Asn-56 to either Gln or Ala resulted in the production of a totally inactive HL which accumulated intracellularly but was not secreted into the culture medium. Therefore, Asn-56 is required for both HL enzyme activity and secretion. The fact that the homologous N-linked glycosylation site (Asn-43) is required for both enzyme activity and secretion for human LPL (Semenkovich et al. 1990. J. Biol. Chem. 265: 5429-5433) indicates that carbohydrate chains at this site are essential for the active conformation and correct folding for secretion of these evolutionarily related lipases. Our observations provide insight into the structural basis of lipase action and secretion.

Animals↗

PubMed1993

Regulation of hepatic triglyceride lipase by thyroid hormone in HepG2 cells.

Hypothyroidism has been reported to be associated with reduced hepatic triglyceride lipase (HTGL) activity. In order to understand the molecular mechanism by which thyroid hormone regulates HTGL activity, effects of triiodothyronine (T3) on HTGL activity, mRNA level, transcription run-on activity, and protein synthetic rate were studied in HepG2 cells. HepG2 cells treated with 1 nM T3 showed an increase in HTGL activity that was first detected at 24 h; HTGL activity continued to increase at 36 h and stayed at the elevated level at 48 and 60 h. At maximal stimulation (48 h), T3-treated cells had the following HTGL activities: 155% in spontaneously released (SR) and 224% in heparin-releasable (HR) HTGL activities (mean levels compared to control). Stimulation of HTGL activity by T3 was dose-dependent and saturable. There was, however, no change in HTGL mRNA level throughout the course of T3 treatment. The effects of T3 were reduced when transcription was blocked by actinomycin D (mean level compared to actinomycin D treatment in the absence of T3: 109% in SR and 127% in HR activities) or translation was blocked by cycloheximide (127% in SR and 122% in HR activities), but HTGL activities were still significantly higher than control. Nuclear run-on assays indicate that T3 did not change the rate of transcription of the HTGL gene. We further determined the rate of HTGL synthesis by measuring the amount of [35S]methionine incorporated into newly synthesized HTGL immunoprecipitated by a monospecific anti-human HTGL antibody. We found that the T3-stimulated increase in HTGL activity was not accompanied by any change in the rate of HTGL biosynthesis. Our experimental data indicate that the T3 stimulation of HTGL activity in HepG2 cells is mediated at posttranscriptional and posttranslational levels. The partial but significant inhibition of the T3 stimulation of HTGL activity by actinomycin D and cycloheximide suggests that the effects of T3 may be mediated by other cellular processes that are more directly regulated by the hormone. This study represents the initial report on the mechanism of HTGL activation by physiological concentrations of thyroid hormone.

Base Sequence↗