Cytotoxic Serine Research Articles

Characterization of a novel, human cytotoxic lymphocyte‐specific serine protease cDNA clone (CSP‐C)**

A human cDNA clone encoding a novel serine protease, cytotoxic serine protease-C(CSP-C), has been isolated from a cDNA library prepared from recombinant interleukin-2 (IL-2)-activated lymphocytes of a patient with a large granular lymphoproliferative disorder. The clone has a 741-base pair open reading frame encoding a putative 246-amino acid protein. The protein sequence contains the catalytic charge relay system characteristic of a serine protease and the conserved N-terminal amino acid sequence of the mature cytotoxic lymphocyte serine proteases found in both mouse and human. The amino acid sequence of CSP-C has 71% identity with the previously reported cytotoxic serine protease-B(CSP-B)/human lymphocyte protease (HLP)/SECT and 57% identity with the granulocyte-specific serine protease cathepsin G. The homology with another lymphocyte-specific serine protease, human Hanukah factor (HF)/Granzyme A was 41%. The transcript is expressed in lymphocytes stimulated with IL-2 or IL-2 plus phytohemagglutinin (PHA). CSP-C is not expressed in B-lymphoblastoid cell lines or in the T-leukemia cell line MOLT4. The cDNA sequence suggests that the protein is expressed as a prepropeptide, as has been found in the other murine and human serine proteases of lymphocyte origin. It has recently been reported that human chromosome 14q11, in addition to containing the genes encoding cytotoxic serine protease B (CSP-B), cathepsin G, and the T-cell receptor alpha and delta genes, also includes an additional genomic DNA clone which cross-hybridized with CSP-B and cathepsin G, cathepsin-like gene-2 (CGL-2). It is likely that the CSP-C cDNA clone reported in this study corresponds to CGL-2.

A cluster of hematopoietic serine protease genes is found on the same chromosomal band as the human alpha/delta T-cell receptor locus.

The chymotrypsin-like family of serine protease genes includes several members that are expressed exclusively in subsets of hematopoietic cells. For example, human neutrophil elastase and cathepsin G are expressed only in myelomonocytic precursors, and cytotoxic-T-cell serine proteases are found only in cytotoxic lymphocytes. We have used a cathepsin G cDNA probe to clone two cathepsin G-like genes (designated CGL-1 and CGL-2) from a human genomic library. We have determined that CGL-1 is identical to a previously identified gene (known as CCPI, CTLA I, or cytotoxic serine protease B) that is expressed only in activated cytotoxic T lymphocytes. We show here that cathepsin G, CGL-1, and CGL-2 are linked on an approximately 50-kilobase locus found on human chromosome 14 at band q11.2. This gene cluster maps to the same chromosomal band as the alpha and delta T-cell receptor genes; this region is involved in most chromosomal translocations and inversions that are specifically associated with T-cell malignancies.

A cytotoxic serine proteinase isolated from mouse submandibular gland

We have isolated a novel cytotoxic factor from the submandibular glands of male BALB/c mice by Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. The cytotoxic factor is a serine proteinase, which belongs to the mouse glandular kallikrein (mGK) family, with an M r of approximately 27000. The purified serine proteinase showed cytotoxic activity against mouse thermocytes in a dose-dependent manner, and a serine proteinase inhibitor, diisopropyl fluorophosphate, blocked its cytotoxic activity.

Genomic organization and chromosomal assignment for a serine protease gene (CSPB) expressed by human cytotoxic lymphocytes

The human cytotoxic serine protease (“granzyme”) B gene has been isolated and sequenced. The gene is approximately 3500 bp in length and consists of five exons and four intervening introns. Its organization therefore conforms closely to that of the previously cloned serine protease genes which are expressed in mouse cytotoxic lymphocytes and rat mast cells. The 5′ upstream region is characterized by an atypical “TATA”-like transcriptional promoter and by a stretch of nucleotides bearing similarity to an enhancer core sequence believed to be requisite for the tissue-specific expression of serine protease genes in the exocrine pancreas. It has previously been demonstrated that mRNA transcripts from the human serine protease B gene are heterogeneous in size, due to frequent faulty intron/exon splicing. Two cryptic splice sites, which are used to generate these aberrant mRNA transcripts, have been identified. Using DNA blot analysis of a panel of human-rodent somatic cell lines, the human serine protease B gene has been localized to chromosome 14.