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N H Lubsen

Publications and source records attributed to N H Lubsen.

At least 19 recordsLinked to original sources
PubMed2001

Lens crystallins and oxidation: the special case of gammaS.

Among lens crystallins, gamma-crystallins are particularly sensitive to oxidation, because of their high amount of Cys and Met residues. They have the reputation to induce, upon ageing, lens structural modifications leading to opacities. A combination of small angle X-ray scattering and chromatography was used to study the oxidation of gamma-crystallins. At pH 7.0, all the gamma-crystallins under study were checked to have the same structure in solution. Under gentle oxidation conditions at pH 8.0, human gammaS (hgammaS) and bovine gammaS (bgammaS) formed disulfide-linked dimers, whereas the other bgamma-crystallins did not. Cys20 was shown to be responsible for dimer formation since the C20S mutant only formed monomers. The hgammaS dimers were stable for weeks and did not form higher oligomers. In contrast, monomeric gammaS-crystallins freshly prepared at pH 8.0, and submitted to more drastic oxidation by X-ray induced free radicals, were rapidly transformed into higher oligomers. So, only extensive oxidation causing partial unfolding could be detrimental to the lens and linked to cataract formation. The gammaS-crystallins lack the temperature-induced opacification observed with the other gamma-crystallins and known as cold cataract. The oxidation-induced associative behaviour and cold cataract are therefore demonstrated to be uncoupled.

Amino Acid Sequence↗
PubMed2001

Association behaviour of human betaB1-crystallin and its truncated forms.

betaB1-crystallin plays an important role in the assembly of betaH-crystallin yet is known to be subject to N-terminal sequence truncations during human lens development and ageing. Here we have over-expressed human betaB1-crystallin, and various truncated forms in Escherichia coli and used mass spectrometry to monitor the monomer molecular weight. Gel permeation chromatography and laser light scattering have been used to estimate the assembly size of the various polypeptides as a function of protein concentration. The full-length betaB1-crystallin behaves as a dimer, like recombinant human betaB2-crystallin, but undergoes further self-association at high protein concentrations, unlike the betaB2-crystallin. Major truncations from the N-terminal extension lead to anomalous behaviour on gel permeation chromatography indicative of altered interactions with the column matrix, whereas light scattering indicated dimers at low protein concentration that self-associate as a function of protein concentration. Loss of 41 residues from the N-terminus, equivalent to an in vivo truncation site, resulted in temperature-dependent phase separation behaviour of the shortened betaB1-crystallin. Good crystals have been grown of a truncated version of human betaB1-crystallin using an in vitro cleavage protocol.

Chromatography, Gel↗
PubMed2000

Gamma S-crystallin of bovine and human eye lens: solution structure, stability and folding of the intact two-domain protein and its separate domains.

Human and bovine gammaS-crystallin (HgammaS and BgammaS) and their isolated N- and C-terminal domains were cloned and expressed as recombinant proteins in E. coli. HgammaS and BgammaS are found to be authentic according to their spectral and hydrodynamic properties. Both full-length proteins and isolated domains are monomeric and exhibit high thermal and pH stabilities. The thermodynamic characterization made use of chemically and thermally-induced equilibrium unfolding transitions at varying pH. In spite of its exemplary two-domain structure, gammaS-crystallin does not show bimodal unfolding characteristics. In the case of BgammaS, at pH 7.0, the C-terminal domain is less stable than the N-terminal one, whereas for HgammaS the opposite holds true. Differential scanning calorimetry confirms the results of chemically-induced equilibrium unfolding transitions. Over the whole pH range between 2.0 and 11.5, HgammaS-crystallin and its isolated domains (HgammaS-N and HgammaS-C) follow the two-state model. The two-state unfolding of the intact two-domain protein points to the close similarity of the stabilities of the constituent domains. Obviously, interactions between the domains do not contribute significantly to the overall stability of gammaS-crystallin. In contrast, the structurally closely related gammaB-crystallin owes much of its extreme stability to domain interactions.

Animals↗
PubMed2000

Molecular basis of a progressive juvenile-onset hereditary cataract.

In a recent paper, patients with a progressive juvenile-onset hereditary cataract have been reported to have a point mutation in the human gammaD crystallin gene (Stephan, D. A., Gillanders, E., Vanderveen, D., Freas-Lutz, D., Wistow, G., Baxevanis, A. D., Robbins, C. M., VanAuken, A., Quesenberry, M. I., Bailey-Wilson, J., et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1008-1012). This mutation results in the substitution of Arg-14 in the native protein by a Cys residue. It is not understood how this mutation leads to cataract. We have expressed recombinant wild-type human gammaD crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in Escherichia coli and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution. Eventually, R14C precipitates. In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers. These data strongly suggest that the observed cataract is triggered by the thiol-mediated aggregation of R14C. The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one. Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities. Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.

Adolescent↗
PubMed2000

Insulin and IGF-I affect the protein composition of the lens fibre cell with possible consequences for cataract.

Explanted newborn rat lens epithelial cells were cultured with various concentrations of FGF-2 and/or insulin or IGF-I for 8-20 days. The accumulation of alphaA-, alphaB-, betaA3/1-, betaB2- and gammaA-F-crystallin was measured. During culture with insulin only, i.e. in the absence of fibre cell differentiation, alphaA- and alphaB-crystallin accumulated to the same level as found in differentiating cells. Culture of epithelial cells with IGF-I led to an increase in alphaB-crystallin, but not in alphaA-crystallin. The addition of insulin under differentiation conditions (in the presence of 25 ng ml(-1)FGF-2) augmented the accumulation of alphaA-crystallin 1.5-fold, the accumulation of betaB2-crystallin two-fold and the accumulation of gammaA-F-crystallin five-fold over that found with FGF-2 only. The accumulation of alphaB- and betaA3/1-crystallin was not affected by insulin in the presence of FGF-2. Adding IGF-I to fibre cells differentiating in the presence of 25 ng ml(-1)FGF-2 resulted in a 1.5-fold increase (of questionable statistical significance) in both alphaA- and alphaB-crystallin and a two to three-fold increase in gammaA-F-crystallin compared to cells cultured with FGF-2 only, no significant effect of IGF-I on the accumulation of betaA3/1- or betaB2-crystallin was found. Comparison of the levels of mRNA and protein suggests that insulin acts to increase the level of transcription. Our results show that the response of fibre cells to insulin or IGF-I differs. Hence, even though half the maximum dosage required for the insulin effect was rather high (between 0.1 and >5 micro g), the effect of insulin cannot be merely transmitted by the IGF-I receptor. Our data further predict that insulin or IGF-I increases the overall ratio of beta- and gamma-crystallin to alpha-crystallin in the fibre cell, which could predispose the lens to cataract.

Animals↗
PubMed1998

Regulation of expression within a gene family. The case of the rat gammaB- and gammaD-crystallin promoters.

The six closely related and clustered rat gamma-crystallin genes, the gammaA- to gammaF-crystallin genes, are simultaneously activated in the embryonic lens but differentially shut down during postnatal development with the gammaB-crystallin gene, the last one to be active. We show here that developmental silencing of the gammaD-crystallin promoter correlates with delayed demethylation during lens fiber cell differentiation. Methylation silencing of the gammaD-crystallin promoter is a general effect and does not require the methylation of a specific CpG, nor does methylation interfere with factor binding to the proximal activator. In later development, the gammaD-crystallin promoter is also shut down earlier by a repressor that footprints to the -91/-78 region. A factor with identical properties is present in brain. Hence, a ubiquitous factor has been recruited as a developmental regulator by the lens. All gamma-crystallin promoters tested contain upstream silencers, but at least the gammaB-crystallin silencer is distinct from the gammaD-crystallin silencer. The gamma-crystallin promoters were found to share a proximal activator (the gamma-box; around -50), which behaves as a MARE. The gammaB-box is recognized with much lower avidity than the gammaD-box. By swapping elements between the gammaB- and the gammaD-crystallin promoter, we show that activation by the gammaB-box requires a directly adjacent -46/-38 AP-1 consensus site. These experiments also uncovered another positive element in the gammaD-crystallin promoter, around -10. In the context of the gammaD-crystallin promoter, this element is redundant; in the context of the gammaB-crystallin promoter, it can replace the -46/-38 element.

Animals↗
PubMed1998

Induction and maintenance of differentiation of rat lens epithelium by FGF-2, insulin and IGF-1.

The differentiation of rat lens epithelial cells to fibre cells can be mimicked using lens epithelial explants, which differentiate in vitro when exposed to fibroblast growth factor (FGF). A previous study demonstrated that FGF is required only for initiation of differentiation: once induced by FGF, differentiation can be maintained by insulin (as assessed by following the accumulation of fibre-cell specific crystallins). The aim of this investigation was to determine whether insulin-like growth factor 1 (IGF-1) can also maintain differentiation and to include a cellular analysis of explants undergoing insulin-or IGF-maintained differentiation in vitro. Measurement of the accumulation of alpha-, beta- and gamma-crystallins showed that IGF-1, like insulin, can replace FGF-2 in directing the pulses of alpha-, beta- and gamma-crystallin gene expression once differentiation is initiated by FGF-2. Cells in both the peripheral and the central region of the explants responded. Immunolocalization of alpha, beta- and gamma-crystallins in these explants showed that a 15 min pulse of FGF-2 triggered the differentiation of only a few cells, whereas a 12 hr pulse primed virtually all the cells for differentiation. This indicates that in explants, individual cells differ in the rate at which they can respond to FGF-2.

Animals↗
PubMed1998

The C-terminal domains of gammaS-crystallin pair about a distorted twofold axis.

The 2-domain gammaS-crystallin, a highly conserved early evolutionary off-shoot of the gamma-crystallin family, is located in the water-rich region of eye lenses. The expressed C-terminal domain, gammaS-C, has been crystallized and the 2.56 A X-ray structure determined. There are two domains in the asymmetric unit which pair about a distorted twofold axis. One of the domains has an altered conformation in a highly conserved region of the protein, the tyrosine corner. The distorted gammaS-C dimer of domains is compared with the highly symmetrical, equivalent recombinant dimer of C-terminal domains from gammaB-crystallin. Sequence changes close to the interface, that distinguish gammaS from the other gamma-crystallins, are examined in order to evaluate their role in symmetrical domain pairing.

Animals↗
PubMed1997

Human alphaB-crystallin. Small heat shock protein and molecular chaperone.

The polymerase chain reaction was used to amplify a cDNA sequence encoding the human alphaB-crystallin. The amplified cDNA fragment was cloned into the bacterial expression vector pMAL-c2 and expressed as a soluble fusion protein coupled to maltose-binding protein (MBP). After maltose affinity chromatography and cleavage from MBP by Factor Xa, the recombinant human alphaB-crystallin was separated from MBP and Factor Xa by anion exchange chromatography. Recombinant alphaB-crystallin was characterized by SDS-polyacrylamide electrophoresis (PAGE), Western immunoblot analysis, Edman degradation, circular dichroism spectroscopy, and size exclusion chromatography. The purified crystallin migrated on SDS-PAGE to an apparent molecular weight (Mr approximately 22,000) that corresponded to total native human alpha-crystallin and was recognized on Western immunoblots by antiserum raised against human alphaB-crystallin purified from lens homogenates. Chemical sequencing, circular dichroism spectroscopy, and size exclusion chromatography demonstrated that the recombinant crystallin had properties similar or identical to its native counterpart. Both recombinant alphaB-crystallin and MBP-alphaB fusion protein associated to form high molecular weight complexes that displayed chaperone-like function by inhibiting the aggregation of alcohol dehydrogenase at 37 degrees C and demonstrated the importance of the C-terminal domain of alphaB-crystallin for chaperone-like activity.

Blotting, Western↗
PubMed1997

Sp1- and octamer-consensus sequence binding proteins during lens fibre differentiation.

The pattern of factors binding to either the Sp1-consensus sequence or to the octamer sequence during in vitro rat lens fibre cell differentiation has been examined by electrophoretic mobility shift assays. With the Sp1-consensus sequence as probe, two major and four minor bands were seen. Three bands were present at all stages of differentiation, two are lost during terminal differentiation and one is present only in late differentiating cells. The octamer probe yielded three bands, which co-migrate with Oct2, Oct3 and Oct7. The exact identity of these factors has not been established. The Oct3-like band was detected only in epithelial cell extracts, the other two bands were also found in fibre cell extracts, whereby the intensity of the Oct2-like band decreased relative to that of the Oct7-like band during differentiation. In transfection studies, a rat gamma D-crystallin promoter in which the proximal activator has been replaced by a dimer of the Sp1-consensus sequence showed a gradual increase in activity with differentiation, in contrast, a similar construct with an octamer dimer was active only during late differentiation and mimicked the pattern of activation of the parental gamma D-crystallin promoter.

Animals↗
PubMed1997

Synergism between temporally distinct growth factors: bFGF, insulin and lens cell differentiation.

Fibroblast growth factors (FGFs) are the only known factors that can induce differentiation of the mammalian lens epithelial cell, while insulin acts only as a mitogen, not as a morphogen. We show here that insulin enhances expression of the alphaA-crystallin gene in lens epithelial cells and induces the synthesis of lens fibre cell specific betaB2- and gamma-crystallins in early differentiated fibre cells. Different signal transduction pathways are required for bFGF or insulin maintained fibre cell differentiation. A 15 min preincubation with bFGF was sufficient for the lens epithelial cells to become competent to undergo insulin maintained differentiation. The phorbol ester TPA could replace bFGF. The bFGF instructed competence to differentiate decays with a half-life of about 30 h. Hence, bFGF and insulin can act in concert to produce a differentiated phenotype even when they are not present simultaneously.

Animals↗
PubMed1997

Mutants of basic fibroblast growth factor identify different cellular response programs.

Mutations, expected to affect the intracellular routing, i.e. additional nuclear localization sequences (NLS; the natural 23 kDa isoform and a 17D27R mutant) and/or a deletion of amino acids 26-29 (23 delta 26-29 and 17 delta 26-29), were introduced in basic fibroblast growth factor (bFGF). The mutants were assayed for their mitotic activity and their capacity to induce a tissue-specific response in human umbilical vein endothelial cells [HUVECs; induction of urokinase plasminogen activator receptor (u-PAR)], or in rat lens epithelial cells (fibre cell differentiation). In HUVECs, the 17D27R mutant had wild type activity, the 23 kDa and the delta 26-29 proteins were impaired in the induction of both mitosis and u-PAR. The delta 26-29 proteins, but not the 23 kDa protein or 17D27R mutant, were also impaired in receptor binding in that they bound only to a subset of receptors. The concentration of 17 kDa bFGF required for half maximal u-PAR response was 30 fold higher than for the half maximal 3H-thymidine incorporation. Addition of an NLS to bFGF strongly inhibited the induction of fibre cell differentiation, though it had little effect on the stimulation of DNA synthesis. The 17 delta 26-29 kDa mutant had wild type differentiation activity but was a poor mitogen for lens epithelial cells.

Animals↗
PubMed1997

Extralenticular expression of Xenopus laevis alpha-, beta-, and gamma-crystallin genes.

PURPOSE: Extralenticular expression of alpha- and beta-crystallin genes has been demonstrated in mammals and expression of gamma-crystallin genes has been shown in Xenopus laevis. To determine a possible correlation between lens determination and crystallin gene expression, the site of expression of (a member of) the alpha-, beta-, and gamma-crystallin gene families was observed before and during lens formation in X. laevis. METHODS: The partial complementary DNAs (cDNAs) of alpha A- and beta A4-crystallin and a gamma-crystallin were cloned from an X. laevis lens cDNA library. The corresponding antisense RNAs were used to analyze the expression of these genes during X. laevis development by wholemount in situ hybridization. RESULTS: Expression of the beta A4- and gamma-crystallin (but not alpha-crystallin) genes could first be detected in the animal cap of the X. laevis gastrula. The beta A4- and gamma-crystallin messengers were also found in the first stage of lens development, when the ectodermal tissue overlying the optic vesicle thickens to form the lens placode. alpha A-crystallin messenger RNAs were only detectable when the lens epithelial cells were formed. CONCLUSIONS: In contrast to observations in most vertebrates, expression of the beta A4- and gamma-crystallin genes was observed to precede that of the alpha A-crystallin gene during lens development of X. laevis, reflecting the determination that in amphibians, the (presumptive) fiber cells are formed before the epithelial cells, whereas in vertebrates, the order is reversed. Expression of beta A4- and gamma-crystallin genes in the ectodermal tissue of the X. laevis gastrula shows that these genes are expressed when this tissue gains competence for lens formation.

Amino Acid Sequence↗
PubMed1996

The mechanism of recruitment of the lactate dehydrogenase-B/epsilon-crystallin gene by the duck lens.

In duck, the housekeeping enzyme lactate dehydrogenase B (LDH-B) and the lens structural protein epsilon-crystallin are encoded by the same single copy gene. Transcription of the gene is initiated from two closely spaced start sites, at -28 and +1. The usage of the downstream site is greatly enhanced in lens. Deletion mapping of the promoter shows that the region -70/+18 specifies the enhanced promoter activity in the lens. A critical role is played by the consensus Sp1 binding site at -50; mutation of this site abolishes lens-preferred expression. Deletion of the +1 transcription initiation site also leads to a decrease in lens-preferred expression, which can be restored by moving the -28 transcription initiation site downstream. By band shift experiments, supershift mobility assays and methylation interference assays, Sp1 was shown to bind to the Sp1 consensus binding site at -50 using either heart or lens nuclear extracts. Co-expression of Sp1 or Sp1-like factors inhibited the activity of an LDH-B/epsilon-crystallin promoter construct by approximately 60% in lens and by 40% in heart cells. Co-expression of Pax-6, a transcription factor shown to be involved in the lens-enhanced expression of a number of other crystallin genes, did not influence the promoter activity of the -130/+650 LDH-B/epsilon-crystallin promoter construct. In contrast to other crystallin promoters, the LDH-B/epsilon-crystallin promoter does not appear to contain a lens-specific element, rather our data lead to a model in which a factor transmitting the effect of Sp1, bound at -50, to the transcription initiation complex is responsible for the lens-preferred expression of the LDH-B/epsilon-crystallin promoter.

Animals↗
PubMed1996

The cooperation between two silencers creates an enhancer element that controls both the lens-preferred and the differentiation stage-specific expression of the rat beta B2-crystallin gene.

The rat beta B2-crystallin gene is active only during a specific stage of the differentiation of rat lens fibre cells directed by basic fibroblast growth factor. The regulatory elements that determine the transient activity of this gene are located in the -750/-123 region and in the first intron. Singly, these elements act as silencers, together they constitute an enhancer that is active only during the specific differentiation stage. An additional silencer is found between -123 and -77. The proximal promoter region contains a Pax-6 binding site at -65/-51. In vitro, binding to this site could be detected but, according to in vivo footprinting experiments, this site is not occupied in the endogenous gene. Furthermore, co-expression of Pax-6 did not enhance promoter activity. Finally, mutation or deletion of this site did not affect promoter activity: the region -37/+10 sufficed for basal promoter activity. The cooperation between the -750/ -123 region and the first intron of the beta B2-crystallin gene not only determines the differentiation stage-specific activity of the gene, but also contributes to the highly increased expression in lens cells compared with non-lens cells.

Amino Acid Sequence↗
PubMed1996

The sequence of regulatory events controlling the expression of the gamma D-crystallin gene during fibroblast growth factor-mediated rat lens fiber cell differentiation.

The transcriptional activation of tissue-specific genes during terminal differentiation must be preceded by the priming of the chromatin and the appearance of the required transacting factors. We have timed these events for the transcriptional activation of the rat gamma D-crystallin gene, a lens fiber cell-specific gene that encodes a structural lens protein, during the (basic fibroblast growth factor (bFGF)-induced) in vitro differentiation of rat lens fiber cells. In vitro, in the presence of bFGF only, the endogenous gamma D mRNA accumulates between Day 10 and Day 15. When insulin is added as well, the differentiation process is accelerated and gamma D mRNA starts to accumulate at Day 8. Demethylation of the gamma D promoter region, as assessed by measuring the methylation state of the ThaI site at -16, occurs much sooner, within 1 day. By genomic footprinting, the first protein interaction with the promoter region was visible at Day 8; full occupancy of the promoter region could be detected only at Day 12. The genomic footprint identified four putative regulatory regions: -141/-131, -88/-71, -55/-45, and -15/-4. Site-directed mutagenesis of the G residues at -55 and -46 resulted in a three- to fivefold decrease in promoter activity of transfected gamma D/CAT reporter genes and also abolished interaction with nuclear extract factor(s). A G-->T mutation at -43 had no effect. The -55/-45 footprint thus derives from a proximal activator. The -88/-71 footprint identifies a silencer of the gamma D promoter in late fiber cell differentiation, as a tetramer of the -85/-67 sequence silenced a tk/CAT construct when transfected into fiber cells at a late stage, but not at an early stage, of in vitro differentiation. To time the appearance of regulatory factors, the activity of a -73/+45 gamma D/CAT (containing the activator region) and of a -1100/+45 gamma D/CAT construct was measured during fiber cell differentiation. The -73/+45 construct was active between Day 5 and Day 14, with a maximum at Day 12. The additional sequence information present in the -1100/+45 construct constrained gamma D promoter activity to between Day 8 and Day 13, with a maximum at Day 10. We conclude that the phased appearance of transacting factors during lens fiber cell differentiation controls the timing of first the activation and then the shutdown of the gamma D-crystallin gene promoter.

Animals↗