Biomedical subjects

J A Udey

Publications and source records attributed to J A Udey.

4 recordsLinked to original sources

PubMed1989

Nonhuman cells correctly sort and process the human lysosomal enzyme cathepsin D.

Cathepsin D, like most lysosomal enzymes, undergoes multiple proteolytic cleavages during its lifetime. Although the significance of the earliest cleavages of cathepsin D is apparent (loss of the NH2-terminal signal peptide and activation peptide), functions of the two later cleavages are not understood and do not occur in all species. To examine these later events, a cDNA coding for human cathepsin D, which is normally processed to a two-chain form, was isolated and then expressed in mammalian cells from species which do not process the enzyme to the two-chain form. Analysis of the expressed human cathepsin D demonstrated proteolytic processing identical with that seen in normal human fibroblasts. Since processing to the two-chain form of the enzyme occurs in the lysosome, these studies revealed that the human cathepsin D was correctly sorted. The data also indicated that the sorting mechanism was conserved between diverse species and that late proteolytic processing in a variety of species was not determined by the presence or absence of the processing enzymes in the cell.

Amino Acid Sequence↗

PubMed1988

Intergenic exchange maintains identity between two human lambda light chain immunoglobulin gene intron sequences.

Evidence for gene conversion or unequal double crossover in the human lambda light chain immunoglobulin locus is presented. The high level of J2C2-J3C3 intron cross-hybridization, the identity of the J lambda and J lambda 3 coding and intron sequences, the presence of multiple base differences between the C lambda 2 and C lambda 3 coding regions, and the presence of both the unconverted and converted alleles in the normal gene pool, suggest that a recombinational event has resulted in the conversion of the J lambda 2 coding and intron sequences to those of J lambda 3 and its flanking sequences. Intergenic exchanges, such as the one described here, may provide a mechanism to maintain sequence homogeneity and functionality among the duplicated members of the human lambda gene family.

Amino Acid Sequence↗

PubMed1987

Human lambda light chain locus: organization and DNA sequences of three genomic J regions.

Evidence for the genomic organization of human lambda light chain joining (J) region gene segments is presented. A mouse J lambda probe was used in Southern hybridizations to localize joining region sequences in a cosmid clone containing the genomic cluster of six human lambda constant (C) region gene segments. The results of these hybridizations suggest the presence of at least one J gene segment upstream from each constant region gene segment. The DNA sequences indicate that the human J lambda 1, J lambda 2, and J lambda 3 gene segments have consensus nonamer and heptamer sequences, proposed to be involved in V-J joining, are capable of encoding the known amino acid sequences for the respective J peptides, and have a sequence which could give a functional RNA splice site at the end of their coding regions. Our data show that a single functional J is located 1.3 or 1.6 kb upstream of each of the C lambda gene segments known to encode the Mcg, Kern- Oz-, and Kern- Oz+ isotypes. Therefore, the gene organization of this region of the human lambda locus is J1C1-J2C2-J3C3. The DNA sequences of J lambda 1, J lambda 2, and J lambda 3 presented in this paper establish that a single J lambda gene segment precedes each expressed C lambda gene segment, and support a model for the evolution of the human lambda JC clusters where J1C1 and J2C2-J3C3 arose from different ancestral JC units.

Animals↗

PubMedDate not supplied

Expression and refolding of recombinant human fibroblast procathepsin D.

Procathepsin D is a precursor of the human lysosomal protease cathepsin D. Due to its short half-life, procathepsin D is difficult to obtain in quantities sufficient to allow structural and enzymatic studies. To obtain large quantities of this precursor, procathepsin D was expressed using the T7 promoter vector pET3a in bacteria that carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lac promoter. At high cell density in rich medium, basal levels of T7 RNA polymerase were sufficient to express recombinant procathepsin D without addition of an exogenous inducer of the lac promoter. The recombinant protein, constituting almost half of the total cell protein, accumulated in intracytoplasmic inclusion bodies and was isolated from the insoluble fraction of lysed cells. Antibodies prepared against the purified recombinant protein were shown to crossreact with native human placental and porcine spleen cathepsin D. Recombinant procathepsin D was solubilized in denaturants and was refolded. After extended preincubation of the denatured protein at acid pH to allow folding and activation of the zymogen, pepstatin inhibitable catalytic proteolysis was detected. These data demonstrated that the glycosylated aspartic protease, procathepsin D can be refolded and activated in an unglycosylated form and thus provides a system for the study of procathepsin D structure and function.

Amino Acid Sequence↗