Biomedical subjects

J M Boschi

Publications and source records attributed to J M Boschi.

4 recordsLinked to original sources

PubMed1994

Novel avian thymic parvalbumin displays high degree of sequence homology to oncomodulin.

CPV3 is a novel avian parvalbumin. It displays an isoelectric point of 4.6, intermediate between that of avian thymic hormone (pI = 4.3) and the muscle parvalbumin isoform (pI = 5.2). Expression of CPV3, like that of avian thymic hormone (ATH), is restricted to the thymic stroma. However, the CPV3 content of chicken thymus tissue (120 micrograms/g tissue) is 4 times lower than that of ATH (500 micrograms/g tissue). The polymerase chain reaction (PCR) was used to gain access to the nucleotide sequence of CPV3. A 147-base pair fragment of the coding sequence, corresponding to residues 48-97, was amplified from total chicken cDNA using degenerate PCR primers. A RACE-PCR strategy was then used to extend the known sequence in both the 5' and 3' directions. The cDNA sequence thus obtained includes 671 base pairs. Primer extension analysis suggests that the cloned cDNA corresponds to a full-length transcript. Northern analysis of chicken mRNA indicates that the average CPV3 transcript is approximately 800 nucleotides in length, significantly smaller than the ATH message (approximately 1000 nucleotides). Southern analysis suggests the presence of a single CPV3 gene in the chicken genome. The translated nucleotide sequence, displaying 108 residues between the initiator and termination codons, is that of a beta-parvalbumin. The CPV3 sequence exhibits 58% identity with ATH and 52% identity with the chicken muscle isoform. Interestingly, CPV3 and the mammalian oncodevelopmental parvalbumin called oncomodulin are identical at 73 of 108 residues (68% identity). Correspondingly, flow-dialysis measurements with 45Ca2+ indicate that the Ca(2+)-binding domains are inequivalent, as in oncomodulin. The apparent dissociation constants, at pH 7.4 in 150 mM NaCl, are approximately 10 nM and 80 nM.

Amino Acid Sequence↗

PubMed1992

Evidence that deprotonation of serine-55 is responsible for the pH-dependence of the parvalbumin Eu3+ 7F0-->5D0 spectrum.

The Eu(III)7F0-->5D0 excitation spectra of the parvalbumins are highly pH-dependent. Below pH 6.0, they exhibit a sharp, partially resolved doublet centered near 5,795 A. However, as the pH is raised, the spectrum becomes increasingly dominated by a much broader signal near 5,784 A. This behavior has been traced to the Eu(III) ion bound at the CD site, but the identity of the moiety undergoing deprotonation remains uncertain. Site-specific mutagenesis studies on the parvalbumin-like protein known as oncomodulin now suggest that the species in question is a liganding serine hydroxyl group. Specifically, replacement of serine-55 by aspartate (the residue present at the corresponding position in the EF site) affords a protein that retains two functional lanthanide binding sites, but fails to undergo the pH-dependent spectral alteration. By contrast, replacement of aspartate-59 by glycine (the corresponding EF site residue) fails to abolish the pH-dependent behavior.

Animals↗

PubMed1991

Interactions between residues in the oncomodulin CD domain influence Ca2+ ion-binding affinity.

Despite striking sequence homology with rat parvalbumin, oncomodulin exhibits much lower affinity for Ca2+ ion. We are attempting to identify the structural basis for this difference by systematically substituting the parvalbumin residue for the oncomodulin residue at points of nonidentity. In this paper, we examine two mutations in the helical segments flanking the CD ion-binding loop. Replacement of Asp-45 in the C helix by lysine, to produce D45K, reduces the dissociation constant for Ca2+ at the CD site from 0.81 to 0.53 microM. Replacement of Lys-69 in the D helix by glycine, to afford K69G, similarly reduces KCa to 0.59 microM. Both mutations perturb the Eu3+ 7Fo----5Do spectral parameters. We also examine the consequences of simultaneous mutations involving positions 57, 59, 60, and 69. Ca(2+)-binding assays and Eu3+ luminescence measurements indicate that there is a conformational interaction between residues 57 and 69 and that this interaction is modulated by residues 59 and 60. When the mutations at positions 57, 59, 60, and 69 are combined, the resulting variant exhibits a KCa value for the CD site of 0.25 microM, reflecting a 3-fold increase in affinity relative to the wild-type protein. Moreover, the pK alpha governing the interconversion of low and high pH forms of the Eu3+ 7Fo----5Do spectrum is increased to 8.1, very close to the value of 8.25 determined previously for rat parvalbumin. In this paper, we also complete our survey of single mutations in the CD loop by examining L58I. Replacement of Leu-58 by isoleucine reduces the affinity of the CD site for Ca2+, raising KCa to 2.2 microM. Finally, we revise our previous estimate of the KCa value for Y57F downward, from 0.80 to 0.64 microM. The earlier result is believed to have been inflated by heterogeneity in the preparation, a consequence of proteolysis.

Amino Acid Sequence↗

PubMed1991

Identification of a novel parvalbumin in avian thymic tissue.

A novel calcium-binding protein has been isolated from chicken thymus tissue. Its molecular weight (approximately 11,500) and characteristic interactions with Tb3+ and Eu3+ identify the protein as a member of the parvalbumin family. Electrophoretically distinct from both chicken (muscle) parvalbumin and avian thymic hormone, it represents the third parvalbumin to be identified in avian tissues and the second to be identified in the avian thymus gland.

Animals↗