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Biomedical subjects

W W De Jong

Publications and source records attributed to W W De Jong.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of two novel human small heat shock proteins: protein kinase-related HspB8 and testis-specific HspB9.

Using search profiles based on the conserved alpha-crystallin domain that is characteristic for small heat shock proteins (sHsps), we traced two new human sHsps. One of these, being the eighth known human sHsp and thus named HspB8, was recently described as a serine-threonine protein kinase (H11), but not identified as an sHsp (C.C. Smith, Y.X. Yu, M. Kulka, L. Aurelian, J. Biol. Chem. 275 (2000)). Northern blotting showed that HspB8/H11 is predominantly transcribed in skeletal muscle and heart, like most other sHsps. The other, named HspB9, is specifically expressed in testis, notably in the spermatogenic cells from late pachytene spermatocyte stage till elongate spermatid stage. While mammalian sHsps are generally highly conserved, mouse HspB9 shows 38% sequence difference with human HspB9, which may confirm its sex-related role.

Amino Acid Sequence↗
PubMed2000

Demyelination and axonal dystrophy in alpha A-crystallin transgenic mice.

Homozygous mice transgenic for alphaA-crystallin, one of the structural eye lens proteins, developed hindlimb paralysis after 8 weeks of age. To unravel the pathogenesis of this unexpected finding and the possible role of alphaA-crystallin in this pathological process, mice were subjected to a histopathological and immunohistochemical investigation. Immunohistochemistry showed large deposits of alphaA-crystallin in the astrocytes of the spinal cord, and in the Schwann cells of dorsal roots and sciatic nerves. Additionally, microscopy showed dystrophic axons in the spinal cord and digestion chambers as a sign of ongoing demyelination in dorsal roots and sciatic nerves. Apart from a few areas with slight alphaA-crystallin-immunopositive structures, the brain was normal. Because the alphaA-crystallin protein expression appeared in specific cells of the nervous system (astrocytes and Schwann cells), the most plausible explanation for the paralysis is a disturbance of cell function caused by the excessive intracytoplasmic accumulation of the alphaA-crystallin protein. This is followed by a sequence of secondary changes (demyelination, axonal dystrophy) and finally arthrosis. In conclusion, alphaA-crystallin transgenic mice develop a peripheral and central neuropathy primarily affecting spinal cord areas at the dorsal side, dorsal root and sciatic nerve.

Animals↗
PubMed1999

Truncation of betaA3/A1-crystallin during aging of the bovine lens; possible implications for lens optical quality.

During aging, extensive modifications of eye lens proteins take place, which may contribute to the development of cataract. Truncation of the accessible extensions of beta-crystallins has been suggested to be an important factor in this process. We therefore studied the truncations of bovine betaA3- and betaA1-crystallin in more detail. These proteins are identical except for the length of their N-terminal extension, 30 and 13 residues, respectively. The water-soluble and -insoluble proteins from cortex and nucleus of bovine lenses of different ages were separated by 2D-gel electrophoresis and immuno-blotted with an antiserum against betaA3. Two major truncation products were detected, which by sequence analysis were found to correspond to betaA3 having lost 11 or 22 amino acids. betaA3(-11) was only observed in the nucleus, whereas betaA3(-22) was present both in cortex and nucleus. We argue, therefore, that each of these two products is produced by a separate proteolytic enzyme. betaA3(-22) can originate by cleavage of betaA3, betaA1 and betaA3(-11). Truncation of betaA3 occurs more readily than that of betaA1, while betaA3(-11) disappears at an intermediate rate. It appears that the longer the N-terminal extension, the easier proteolysis takes place. Truncated proteins are not necessarily prone to end up in the water-insoluble fractions; other modifications leading to charge changes are more likely to be responsible for insolubilization. Truncation of the extensions of beta-crystallins could be a functional rather than a harmful process during aging of the lens; by modulating protein repulsion, it may help to maintain the protein concentration gradient that is necessary for the optical quality of the lens.

Aging↗
PubMed1998

HspB3, the most deviating of the six known human small heat shock proteins.

From the alignment of 14 EST clones, the cDNA sequence of a novel human small heat shock protein (sHsp), called HspB3, could be deduced. The 3' part of the HspB3 cDNA is 99% identical to that of the previously reported HspL27 cDNA (W.Y. Lam, S.K. Wing Tsui, P.T. Law, S.C. Luk, K.P. Fung, C.Y. Lee, M.M. Waye, Isolation and characterization of a human heart cDNA encoding a new member of the small heat shock protein family-HSPL27, Biochim. Biophys. Acta 1314 (1996) 120-124). We argue that the HspB3 cDNA sequence is a corrected version of the HspL27 cDNA. The HspB3 cDNA is 742 bp long and contains an open reading frame specifying a polypeptide of 150 amino acid residues. Among the six known human sHsps it is evident that HspB3 is the most deviating one, having a unique N-terminal domain and essentially lacking a C-terminal extension. Northern blot analysis shows that in smooth muscle tissue the cDNA hybridizes with mRNA of about 0.9 kb.

Amino Acid Sequence↗
PubMed1995

The mutation Asp69-->Ser affects the chaperone-like activity of alpha A-crystallin.

alpha-Crystallins are members of the family of small heat-shock proteins. The conformation and mode of action of these 'junior chaperones' are unknown. To investigate the structure and chaperone-like activity, four mutants of bovine alpha A-crystallin were generated by site-directed mutagenesis. In comparison with wild-type alpha A-crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat-protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat-protective capacity were observed. Although it is assumed that small heat-shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in alpha-crystallin can also influence the efficiency of substrate binding.

Amino Acid Sequence↗
PubMed1993

Comparison of stability properties of lactate dehydrogenase B4/epsilon-crystallin from different species.

epsilon-Crystallin occurs as an abundant lens protein in many birds and in crocodiles and has been identified as heart-type lactate dehydrogenase (LDH-B4). Lens proteins have, due to their longevity and environmental conditions, extraordinary requirements for structural stability. To study lens-protein stability, we compared various parameters of LDH-B4/epsilon-crystallin from lens and/or heart of duck, which has abundant amounts of this enzyme in its lenses, and of chicken and pig, which have no epsilon-crystallin. Measuring the thermostability of LDH-B4 from the different sources, the t50 values (temperature at which 50% of the enzyme activity remains after a 20-min period) for LDH-B4 from duck heart, duck lens and chicken heart were all found to be around 76 degrees C, whereas pig heart LDH-B4 was less thermostable, having a t50 value of 62.5 degrees C. A similar tendency was found with urea inactivation studies. Plotting the first-order rate constants obtained from inactivation kinetic plots against urea concentration, it was clear that LDH-B4 from pig heart was less stable in urea than the homologous enzymes from duck heart, chicken heart and duck lens. The duck and chicken enzymes were also much more resistant against proteolysis than the porcine enzyme. Therefore, it is concluded that avian LDH-B4 is structurally more stable than the homologous enzyme in mammals. This greater stability might make it suitable to function as a crystallin, as in duck, but is not necessarily associated with high lens expression, as in chicken.

Amino Acid Sequence↗
PubMed1992

Expression and aggregation of recombinant alpha A-crystallin and its two domains.

The 20 kDa alpha A and alpha B subunits of alpha-crystallin from mammalian eye lenses form large aggregates with an average molecular weight of 800,000. To get insight into the interactions responsible for aggregate formation, we expressed in Escherichia coli the putative N- and C-terminal domains of alpha A-crystallin, as well as the intact alpha A-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20-60% of total protein). Recombinant alpha A-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant alpha A-crystallin forms aggregates comparable with alpha-crystallin aggregates from calf lenses, whereas the C-terminal domain forms dimers or tetramers. The N-terminal domain is expressed in an initially water-insoluble form. After solubilization, denaturation and reaggregation the N-terminal domain exists in a high molecular weight multimeric form. These observations suggest that the interactions leading to aggregation of alpha A-crystallin subunits are mainly located in the N-terminal half of the chain.

Base Sequence↗
PubMed1990

Differential synthesis of crystallins in the developing rat eye lens.

The patterns of protein synthesis in rat lenses ranging in age from newborn to 4 months were compared. After incubation of lenses in [35S]methionine-containing medium it was possible to identify the de novo synthesized crystallins by two-dimensional gel electrophoresis and fluorography, in combination with peptide mapping and immunoblotting. It was found that the relative synthesis of alpha A and beta A3 stays fairly constant in rat lenses of all investigated ages. The relative synthesis of beta B2 and gamma s shows a pronounced increase with age in these post-natal lenses. A differential decrease can be observed in the relative synthesis of the other six gamma-crystallins (gamma A-gamma F). There appears to be a good correlation between the changes in relative synthesis of the various crystallins and previously reported alterations in mRNA levels, although certain mRNAs exhibit marked differences in translational efficiency.

Aging↗
PubMed1984

Interaction of crystallins with the cytoskeletal-plasma membrane complex of the bovine lens.

The isolated lenticular plasma membrane-cytoskeleton complex, when analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis, shows reproducibly a significant amount of crystallins, mainly alpha-crystallin. Evidence is provided that purified plasma membranes from the bovine lens also associate selectively with a limited number of newly synthesized polypeptides on cell-free translation of calf lens polyribosomes and addition of the membranes to the incubation medium. This capability is retained by purified lens membrane junctions. The polypeptides that are selected comprise alpha-crystallin chains (in particular alpha A2-crystallin), actin, vimentin and beta B1a-crystallin. Sequence analysis revealed that the latter has in its N-terminal extension a characteristic Pro-Ala track. The designation 'PAPA-arm' is proposed for this N-terminal region, comprising the alternating Pro-Ala sequence, that has previously also been found in rabbit myosin and might be responsible for anchoring beta B1a-crystallin to lens membranes.

Amino Acid Sequence↗
PubMed1983

Aggregation behavior of the bovine beta-crystallin Bp chain studied by limited proteolysis.

The bovine beta-crystallin Bp chain is organized into two very similar domains, with short extensions at both N- and C-termini, and two alternative models for the beta Bp dimer have been proposed (Wistow, G., Slingsby, C., Blundell, T., Driessen, H.P.C., De Jong, W.W. and Bloemendal, H. (1981) FEBS Lett. 133, 9-16). By limited proteolysis the C-terminal arms can be cleaved off rapidly from the beta Bp dimer, while the N-terminal arms are more difficult to remove. Trypsin divides the beta Bp chain into two fragments which approximately correspond to the two structural domains. Dissociation and reassociation of the different products of limited proteolysis indicated that: the C-terminal arm extends freely from the surface and is not involved in subunit-contact; at least one N-terminal arm seems required for dimer formation; the N-terminal domains have a greater tendency to associate than the C-terminal domains and, when mixed, the purified domains reassociate partially to a Mr 50 000 structure like native beta Bp. These findings support the more extended dimer model of beta Bp.

Animals↗
PubMed1977

Primary structures of alpha-crystallin A chains of elephant, whale, hyrax and rhinoceros.

As part of a study of the evolutionary development of the eye lens protein alpha-crystallin the 173-residue A chain of this protein has been studied in elephant, whale, hyrax and rhinoceros. The primary structures were inferred mainly from amino acid compositions of peptides obtained by enzymic digestions and CNBr cleavage. The positions of substitutions, as compared to the known bovine A chain, were confirmed by Edman degradation. In accordance with the previously observed slow rate of evolution of the A chain only a small number of substitutions was found among these species. Elephant and hyrax share a number of unique substitutions, strongly indicating a common ancestry of these two species within the mammalian class.

Amino Acid Sequence↗
PubMed1976

The amino-acid sequence of the alpha-crystallin A chains of red kangaroo and Virginia opossum.

The amino acid sequence of the A chain of the eye lens protein alpha-crystallin from the red kangaroo (Macropus rufus) was completely determined by manual Edman degradation of tryptic, thermolytic and cyanogen bromide peptides. The sequence of the alpha-crystallin A chain from the Virginia opossum (Didelphis marsupialis) was deduced from amino acid analyses and partial Edman degradation of peptides. The 173-residue A chains of kangaroo and opossum differ in six positions, whereas comparison with the bovine alpha-crystallin A chain reveals 17 and 22 substitutions, respectively. Most substitutions occur in the COOH-terminal part of the chain.

Amino Acid Sequence↗
PubMed1976

Model studies of enzymatic NH2-terminal acetylation of porteins with des-Nalpha1-acetyl-alpha-melanotropin as a substrate.

The present study describes the acetylation by an enzyme present in calf lens of a synthetic tridecapeptide [analogous to alpha-melanotropin (alpha-melanocyte stimulating hormone) but lacking the naturally occurring NH2-terminal acetyl group: des-Nalpha1-Ac-alpha-melanotropin]. The reaction is specific for the alpha-amino group of the NH2-terminal amino acid. The minimum length required for the substrate to become acetylated appears to be a sequence of five to eight amino acid residues. Modification of the internal lysine decreases the incorporation of acetate, irrespective of the size of the blocking group.

Acetates↗
PubMed1975

Hemoglobin Koya Dora: high frequency of a chain termination mutant.

Approximately 10% of the members of the Koya Dora tribe from Andhra Pradesh (India) carry an alpha chain hemoglobin variant, Hb Koya Dora (Hb KD), usually in amounts of 0.5%-2% of total hemoglobin. In four presumed homozygotes for Hb KD, up to 10% of the abnormal hemoglobin was present. The alpha chain of Hb KD was found to be elongated by at least 16 residues, possibly as a result of a mutation of the normal alpha chain termination codon UAA TO UCA, coding for serine. A pedigree in which two individuals possess Hb KD as well as the alpha chain variant Hb Rampa and normal Hb A proves the existence of two alpha chain loci in this population. Hb DK resembles the previously described Hb Constant Spring [6, 7] in many aspects, probably also in its alpha thalassemia-like expression.

Amino Acids↗