spacer
2. Authors Pham CT. Ley TJ.
Title The role of granzyme B cluster proteases in cell-mediated cytotoxicity. [Review] [55 refs]
Source Seminars in Immunology. 9(2):127-33, 1997 Apr.
Abstract Granzymes are neutral serine proteases that are stored in the specialized lytic granules of cytotoxic lymphocytes. A mutation introduced into the granzyme B locus leads to a severe defect in the ability of cytotoxic lymphocytes to induce apoptosis in susceptible target cells, and reduces the severity of class I-dependent acute graft-versus-host disease (GvHD). However, granzyme B-independent cytotoxicity also exists: in CD8+ cells, most of it is perforindependent, but in CD4+ cells, the Fas system and an additional pathway are involved. The identification of these pathways and their physiological relevance may lead to new approaches for inhibiting cytotoxic lymphocyte functions. [References: 55]

spacer
3. Authors Pham CTN. Armstrong RJ. Zimonjic DB. Popescu NC. Payan DG. Ley TJ.
Title Molecular cloning, chromosomal localization, and expression of murine dipeptidyl peptidase I.
Source Journal of Biological Chemistry. 272(16): 10695-703, 1997 Apr 18.
Abstract Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease that catalyzes the sequential removal of dipeptides from the amino termini of various protein substrates. We have isolated a cDNA coding for murine DPPI from mouse thymus and spleen cDNA libraries. The deduced amino acid sequence codes for a protein of 462 amino acid residues; comparison of this deduced sequence with that of rat and human DPPI revealed 90.1% and 77.8% identity, respectively. Using DPPI cDNA, we obtained two BAC (Bacterial Artificial Chromosome) clones that contained the murine DPPI locus. The DPPI gene consists of seven exons and 6 introns, and spans approximately 20 kilobases. Using fluorescence in situ chromosome hybridization, we localized murine DPPI to chromosome 7D3-E1.1. We determined that DPPI protein is widely distributed in mouse tissues, although its relative abundance varies from tissue to tissue. In contrast to previous reports, we show here that DPPI mRNA and protein levels and enzymatic activity are unchanged during in vitro T cell activation, implying that this enzyme is not rate-limiting for granzyme processing.

spacer
4. Authors Karen W. Makar,* Christine T. N. Pham,** Marlin H. Dehoff,* Siobhan M. O'Connor,** Susan M. Jacobi,** and V. Michael Holers*
Title An Intronic Silencer Regulates B Lymphocyte Cell- and Stage-Specific Expression of the Human Complement Receptor Type 2 (CR2, CD21) Gene
Source Journal of Immunology, 1998, 160:1268-1278.
Abstract Human CR2 (CD21) is a B lymphocyte protein whose surface expression is restricted primarily to the mature cell stage during development. To study the transcriptional mechanisms that govern cell- and stage-restricted CR2 expression, we first performed transient transfection analysis using constructs extending from-5 kb to +75 bp (-5 kb/+75) in the CR2 promoter. The promoter was found to be broadly active, with no evidence of cell- or stage-specific reporter gene expression. However, the addition of a 2.5-kb intronic gene segment (containing a DNase I hypersensitive site) to the (-5-kb/+75) construct resulted in appropriate reporter gene expression, defined as the silencing of the (-5-kb/+75) promoter activity only in non-CR2-expressing cells. Interestingly, appropriate reporter gene expression required stable transfection of the constructs in cell lines, suggesting nuclear matrix or chromatin interactions may be important for appropriate CR2 gene expression. Importantly, transgenic mice also required the intronic silencer to generate lymphoid tissue-specific reporter gene expression. Some transgenic founder lines did not demonstrate reporter gene expression, however, indicating that additional transcriptional regulatory elements are present in other regions of the CR2 gene. In summary, these data support the hypothesis that human CR2 expression is regulated primarily by an intronic silencer with lineage- and B cell stage-specific activity. The Journal of Immunology, 1998,160: 1268-1278.

spacer
5. Authors Pham CTN. Thomas DA. Mercer JD. Ley TJ.
Title Production of fully active recombinant murine granzyme B in yeast
Source Journal of Biological Chemistry. 273 (3):1629-1633, 1998 Jan 16.
Abstract Granzyme B (GzmB) is a neutral serine protease found in cytotoxic lymphocytes; this enzyme is critically involved in delivering the rapid apoptotic signal to susceptible target cells. GzmB has been difficult to study and has not yet been produced in non-mammalian systems because of the complex processing events that are thought to be required for its activation. In this report, we have successfully produced fully active, soluble recombinant GzmB (rGzmB) in a yeast-based system by fusing GzmB cDNA in frame with yeast alpha-factor cDNA, using the yeast KEX2 signal peptidase to release the processed enzyme into the supernatant of yeast cultures. We expressed the proenzyme form of GzmB as well and determined that pro-GzmB is efficiently converted to its active form by the cysteine proteinase dipetidyl peptidase I. the fully processed enzyme was able to hydrolyze the synthetic substrate N-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-asparyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a k(cat) of 17 s(-1) and catalytic efficiency k(cat)/K-m of 181,237 M-1 s(-1); the recombinant enzyme is therefore at least twice as active as purified native GzmB. In addition, the recombinant enzyme hydrolyzes Boc-Ala-Ala-Met thiobenzyl ester with a K-cat of 3.2 s(-1) and a catalytic efficiency k(cat)/K-m of 65,306 M-1 s(-1). Purified rGzmB can also cleave the putative substrate caspase-3 into its signature p20/p10 forms. Unlike caspases, rGzmB is not sensitive to inhibition by several peptide-based inhibitors, including Ac-DEVD-CHO, Ac-YVAD-CMK, and ZIETD-FMK as well as Zn2+ (a known inhibitor of caspase-3). Structural studies of rGzmB may allow us to better understand the substrate specificity of this enzyme and to design better inhibitors.