Molecular cloning of seprase: a serine integral membrane protease from human melanoma

Abstract

Seprase is a homodimeric 170 kDa integral membrane gelatinase whose expression correlates with the invasiveness of the human melanoma cell line LOX. Here, we report the molecular cloning of a cDNA that encodes the 97 kDa subunit of seprase. Its deduced amino acid sequence predicts a type II integral membrane protein with a cytoplasmic tail of 6 amino acids, followed by a transmembrane domain of 20 amino acids and an extracellular domain of 734 amino acids. The carboxyl terminus contains a putative catalytic region (approximately 200 amino acids) which is homologous (68% identity) to that of the nonclassical serine protease dipeptidyl peptidase IV (DPPIV). The conserved serine protease motif G-X-S-X-G is present as G-W-S-Y-G. However, sequence analysis of seprase cDNA from LOX and other cell lines strongly suggests that seprase and human fibroblast activation protein alpha (FAP alpha) are products of the same gene. We propose that seprase/FAP alpha and DPPIV represent a new subfamily of serine integral membrane proteases (SIMP).

A Novel Protease-docking Function of Integrin at Invadopodia

“…2A) or collagen or gelatin (Fig. 2, B and C) with mAbs D8 or D28 to detect seprase (14,15,18) and C27 to detect ␤ 1 integrin. In three independent experiments, a stable association of integrin and seprase was not detected when cells were cultured on plastic ( Fig.…”

“…Antibodies and Immunofluorescence Labeling-Anti-seprase monoclonal antibodies (mAbs) D28 and D8 have previously been described (14,15). Anti-␤ 1 polyclonal (number 3847) and mAb 13 antibodies were used to detect ␤ 1 integrins (17), and anti-vitronectin receptor antibodies was used to detect the ␤ 3 subunit of the vitronectin receptor (Life Technologies, Inc.).…”

“…We have shown that in LOX melanoma cells, a 170-kDa membrane gelatinase, seprase, was localized to invadopodia and associated with the invasive phenotype (13)(14)(15)(16). Sequencing data on the 97-kDa protein subunit of seprase indicates only a short (six) amino acid sequence at the cytoplasmic amino terminus (14), suggesting that seprase localization at invadopodia may be dependent upon other membrane proteins such as integrins. Here, we show immunoprecipitation, immunofluorescence, and cell surface cross-linking experiments demonstrating that seprase and ␣ 3 ␤ 1 integrin associate at invadopodia in a collagendependent manner.…”

“…Then, the samples were immediately centrifuged at 25°C. The filtrates were subjected to immunoblotting and gelatin zymography as described (14,15). Antigens were essentially absent from post-sonication beads.…”

“…Moreover, both adhesive and signaling activities of integrins can be regulated by the interaction between integrins and the urokinase plasminogen activator/receptor (12). We have shown that in LOX melanoma cells, a 170-kDa membrane gelatinase, seprase, was localized to invadopodia and associated with the invasive phenotype (13)(14)(15)(16). Sequencing data on the 97-kDa protein subunit of seprase indicates only a short (six) amino acid sequence at the cytoplasmic amino terminus (14), suggesting that seprase localization at invadopodia may be dependent upon other membrane proteins such as integrins.…”