The proto-oncogene c-KIT encodes a growth factor receptor, KIT, with ligand-dependent tyrosine kinase activity that is expressed by several cell types including mast cells. c-KIT juxtamembrane coding region mutations causing constitutive activation of KIT are capable of transforming cell lines and have been identified in a human mast cell line and in situ in human gastrointestinal stromal tumors, but have not been demonstrated in situ in neoplastic mast cells from any species. To determine whether c-KIT juxtamembrane mutations occur in the development of mast cell neoplasms, we examined canine mastocytomas, which are among the most common tumors of dogs and which often behave in a malignant fashion, unlike human solitary mastocytomas. Sequencing of c-KIT cDNA generated from tumor tissues removed from seven dogs revealed that three of the tumors contained a total of four mutations in an intracellular juxtamembrane coding region that is completely conserved among vertebrates. In addition, two mutations were found in three mast cell lines derived from two additional dogs. One mutation from one line matched that found in situ in one of the tumors. The second was found in two lines derived from one dog at different times, indicating that the mutation was present in situ in the animal. All five mutations cause high spontaneous tyrosine phosphorylation of KIT. Our study provides in situ evidence that activating c-KIT juxtamembrane mutations are present in, and may therefore contribute to, the pathogenesis of mast cell neoplasia. Our data also suggest an inhibitory role for the KIT juxtamembrane region in controlling the receptor kinase activity.

Tryptases are serine proteases implicated in asthma and are very highly expressed in human mast cells. They fall into two groups, ␣ and ␤. Although several related tryptase mRNAs are known, it is unclear which if any are transcripts of separate haploid genes. The studies described here investigated the nature and number of human tryptases and sought possibly novel members of the family. To this end, two human bacterial artificial chromosome (BAC) clones containing tryptase genes were identified and mapped to chromosome 16p13.3, of which ϳ2.2 megabases are syntenic with the part of mouse chromosome 17 containing tryptase genes mouse mast cell protease (mMCP)-6 and -7. Sequencing and restriction mapping suggest that the BACs may partially overlap. Sequenced BAC genes correspond to three known ␤-tryptases (␤I, ␤II, and ␤III), an ␣-like gene, and a pair of novel hybrid genes related partly to ␣/␤-tryptases and partly to orthologs of mMCP-7. ␤II and ␤III, ␤I and ␣II, as well as the two mMCP-7-like genes, may be alleles at single loci; in total, there are at least three nonallelic tryptase genes in the isolated BAC clones. DNA blotting and restriction analysis suggest that the BACs include most members of the immediate tryptase family. Thus, chromosome 16p13.3 harbors a cluster of known and previously undescribed members of the tryptase gene family.

Previously, this laboratory identified clusters of α-, β-, and mast cell protease-7-like tryptase genes on human chromosome 16p13.3. The present work characterizes adjacent genes encoding novel serine proteases, termed γ-tryptases, and generates a refined map of the multitryptase locus. Each γ gene lies between an α1H Ca2+ channel gene (CACNA1H) and a βII- or βIII-tryptase gene and is ∼30 kb from polymorphic minisatellite MS205. The tryptase locus also contains at least four tryptase-like pseudogenes, including mastin, a gene expressed in dogs but not in humans. Genomic DNA blotting results suggest that γI- and γII-tryptases are alleles at the same site. βII- and βIII-tryptases appear to be alleles at a neighboring site, and αII- and βI-tryptases appear to be alleles at a third site. γ-Tryptases are transcribed in lung, intestine, and in several other tissues and in a mast cell line (HMC-1) that also expresses γ-tryptase protein. Immunohistochemical analysis suggests that γ-tryptase is expressed by airway mast cells. γ-Tryptase catalytic domains are ∼48% identical with those of known mast cell tryptases and possess mouse homologues. We predict that γ-tryptases are glycosylated oligomers with tryptic substrate specificity and a distinct mode of activation. A feature not found in described tryptases is a C-terminal hydrophobic domain, which may be a membrane anchor. Although the catalytic domains contain tryptase-like features, the hydrophobic segment and intron-exon organization are more closely related to another recently described protease, prostasin. In summary, this work describes γ-tryptases, which are novel members of chromosome 16p tryptase/prostasin gene families. Their unique features suggest possibly novel functions.

In prior work we showed that a metallogelatinase is secreted from dog mastocytoma cells and directly activated by exocytosed mast cell ␣-chymase. The current work identifies the protease as a canine homologue of progelatinase B (92-kDa gelatinase, MMP-9), determines the sites cleaved by ␣-chymase, and explores the regulation of gelatinase expression in mastocytoma cells. To obtain a cDNA encoding the complete sequence of mastocytoma gelatinase B, a 2.3-kilobase clone encoding progelatinase was isolated from a BR mastocytoma library. The sequenced cDNA predicts a 704-amino acid protein 80% identical to human progelatinase B. Regions thought to be critical for active site latency, such as the Cys-containing propeptide sequence, PRCGVPD, and the catalytic domain sequence, HEFGHALGLDHSS, are entirely conserved. Cleavage of progelatinase B by purified dog ␣-chymase yielded an ϳ84-kDa product that contained two NH 2 -terminal amino acid sequences, QTFEGDLKXH and EGDLKXHHND, which correspond to residues 89 -98 and 92-101 of the cDNA predicted sequence, respectively. Thus, ␣-chymase cleaves the cata- 1 family, gelatinase B must first be processed to an active form before it can degrade its preferred matrix substrates. Whether initiated by reagents such as chaotropes, oxidants, or proteases, MMP activation proceeds along a putative common pathway which involves disruption of an intramolecular interaction between a propeptide Cys and Zn 2ϩ in the active site, a mechanism which has been termed the cysteine switch (8).Gelatinase B, like other MMP's, appears to be biologically ubiquitous. It has been identified in all major organ systems and implicated in numerous homeostatic and pathological processes. Wound injury models suggest a unique role for the enzyme in remodeling of basement membranes, which are composed mainly of collagen IV, its principal collagenous substrate (9 -12). While mechanisms regulating MMP activation in vivo remain unclear, activation pathways involving proteases are likely. Proteolytic cleavage on the COOH-terminal side of the propeptide domain sequence, PRCGVPD, disrupts the cysteine switch and permits further zymogen processing by autocatalytic cleavages. Autolysis truncates the proenzyme at both the NH 2 and COOH termini to yield enzymatically active forms (8). Serine proteases, such as plasmin and furin (13-15), as well as certain members of the MMP family (14, 16 -18) are among the proposed physiologic activators of pro-MMP's.Mast cells are widespread, extravascular mononuclear cells which release serine proteases during degranulation. They produce and store tryptic and chymotryptic enzymes (tryptases and chymases, respectively) which have been implicated in MMP activation pathways (19 -24). We previously reported that BR dog mastocytoma cells constitutively release a 92-kDa gelatinolytic protease similar to gelatinase B, but secrete its activator, ␣-chymase, only in response to a degranulating stimulus (24). Those data predicted that activation of gelatinase by ␣-chymase would occur in the sett...

Genetic alpha-tryptase deficiency is common and varies strikingly between ethnic groups. Because beta-tryptases are implicated in allergic disorders, inherited differences in alpha/beta-genotype may affect disease susceptibility, severity and response to tryptase inhibitor therapy.