The amplified mouse squamous cell carcinoma antigen gene locus contains a serpin (Serpinb3b) that inhibits both papain-like cysteine and trypsin-like serine proteinases $ David J. Askew, a Yuko S. Askew, a Yukari Kato, a Cliff J. Luke, a Stephen C. Pak, a Dieter Bro ¨mme, b and Gary A. Silverman a, * a Department of Pediatrics and the Division of Newborn Medicine, Harvard Medical School, Children’s Hospital, 300 Longwood Avenue, Enders 9, Boston, MA 02115, USA b Department of Human Genetics, Mount Sinai School of Medicine, Fifth Avenue at 100th Street, New York, NY 10029, USA Received 29 January 2004; accepted 31 January 2004 Available online 14 April 2004 Abstract The clade B serpins occupy a unique niche among a larger superfamily by predominantly regulating intracellular proteolysis. In humans, there are 13 family members that map to serpin gene clusters at either 6p25 or 18q21. While most of these serpins display a unique inhibitory profile and appear to be well conserved in mammals, the clade B loci of several species show evidence of relatively recent genomic amplification events. However, it is not clear whether these serpin gene amplification events yield paralogs with functional redundancy or, through selective pressure, inhibitors with more diverse biochemical activities. A recent comparative genomic analysis of the mouse clade B cluster at 1D found nearly complete conservation of gene number, order, and orientation relative to those of 18q21 in humans. The only exception was the squamous cell carcinoma antigen (SCCA) locus. The human SCCA locus contains two genes, SERPINB3 (SCCA1) and SERPINB4 (SCCA2), whereas the mouse locus contains four serpins and three pseudogenes. At least two of these genes encoded functional, dual cross-class proteinase inhibitors. Mouse Serpinb3a was shown previously to inhibit both chymotrypsin-like serine and papain-like cysteine proteinases. We now report that mouse Serpinb3b extends the inhibitory repertoire of the mouse SCCA locus to include a second cross-class inhibitor with activity against both papain-like cysteine and trypsin-like serine proteinases. These findings confirmed that the genomic expansion of the clade B serpins in the mouse was associated with a functional diversification of inhibitory activity. D 2004 Elsevier Inc. All rights reserved. Keywords: Serpins; Proteinases; Chromosome 18; Cathepsins; Evolution The serpins 1 are a superfamily of serine and cysteine proteinase inhibitors [1,2]. There are now well over 700 family members deposited in nucleotide and protein data- bases. The large number of serpin genes is due to their distribution within all of the major domains of life as well as extensive local amplifications within different species [1,2]. The key to serpin activity is the reactive site loop (RSL) domain located near the carboxy terminus. This motif serves as ‘‘bait’’ for a target proteinase with a single amino acid at the reactive center (P1 residue) as the primary (but not exclusive) determinant of target specificity [3–6]. Different serpin families have evolved by inter- and intrachromosomal amplification events followed by RSL diversification [7–9]. Comparative genomic analysis pro- vides insight into the extent of these amplification events. 0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2004.01.014 $ Sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AY367774 (Serpinb3b), AY367775 (Serpinb3c), and AY367776 (Serpinb3d). * Corresponding author. Fax: (617) 731-8962. E-mail address: gary.silverman@childrens.harvard.edu (G.A. Silverman). www.elsevier.com/locate/ygeno 1 Abbreviations used: serpin, serine proteinase inhibitor; SCCA, squamous cell carcinoma antigen; RSL, reactive site loop; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transfer- ase; cat, cathepsin; CMA1, mast cell chymase; HNE, human neutrophil elastase; uPA, urokinase-type plasminogen activator; Succ-AAPF-pNA, succinyl-Ala-Ala-Pro-Phe-para-nitroanilide; MeO-Succ-AAPV-pNA, methoxy-Succ-Ala-Ala-Pro-Val-pNA; VLK-pNA, d-Val-Leu-Lys-pNA; EGR-pNA, H-Glu-Gly-Arg-pNA; (Z-PR) 2 -R110, (Z-Pro-Arg) 2 -R110; (Z- FR) 2 -R110, (Z-Phe-Arg) 2 -R110; SI, stoichiometry of inhibition; MALDI- MS, matrix-associated laser desorption ionization mass spectroscopy. Genomics 84 (2004) 166 – 175