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Membrane-type serine protease 1 (MT-SP1) was recently cloned, and we now report its biochemical characterization. MT-SP1 is predicted to be a type II transmembrane protein with an extracellular protease domain. This localization was experimentally verified using immunofluorescent microscopy and a cell-surface biotinylation technique. The substrate specificity of MT-SP1 was determined using a positional scanning-synthetic combinatorial library and substrate phage techniques. The preferred cleavage sequences were found to be (P4-(Arg/Lys)P3-(X)P2-(Ser)P1-(Arg)P1-(Ala)) and (P4-(X)P3-(Arg/Lys)P2-(Ser)P1(Arg) P1(Ala)), where X is a non-basic amino acid. Protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator are proteins that are localized to the extracellular surface and contain the preferred MT-SP1 cleavage sequence. The ability of MT-SP1 to activate PARs was assessed by exposing PAR-expressing Xenopus oocytes to the soluble MT-SP1 protease domain. The latter triggered calcium signaling in PAR2-expressing oocytes at 10 nM but failed to trigger calcium signaling in oocytes expressing PAR1, PAR3, or PAR4 at 100 nM. Singlechain urokinase-type plasminogen activator was activated using catalytic amounts of MT-SP1 (1 nM), but plasminogen was not cleaved under similar conditions. The membrane localization of MT-SP1 and its affinity for these key extracellular substrates suggests a role of the proteolytic activity in regulatory events.

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a ''fold-specific'' inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription-PCR with PC-3 poly(A)؉ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsinfold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membranetype serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Table A3. Bond lengths [A] and angles [ 0 for [Co(3-Cl-acacen)(NH3)2]BPh4. Co-O (1) #1 Co-N(1) #1#1 C(S)-H(SA) C(S)-H(5C) C(6)-H(6B) C(20)-C(21) C(20)-B C(21)-H(21) C(22)-H(22) C(23)-H(23) C(24)-H(24) C(40)-C(41)#1 C(40)-B C(41)-H(41) C(42)-H(43) C(43) -H(43) C(30)-C(31) C(31)-C(32) C(32)-C(33) C(33) -C(32)#1 C(5)-H(5B) 0.96 C(6)-H(6A) 0.96 C(6)-H(6C) 0.96 C(20)-C(25) 1.407(8) C(21)-C(22) 1.381(9) C(22)-C(23) 1.362(10) C(23)-C(24) 1.364(10) C(24)-C(25) 1.409(9) C(25)-H(25) 0.93 C(40)-C(41) 1.395(7) C(41)-C(42) 1.392(9) C(42)-C(43) 1.373(8) C(43)-C(42)#1 1.373(8) C(30)-C(31)#1 1.389(7) C(30)-B 1.629(13) C(31)-H(31) 0.93 C(32)-H(32) 0.93 C(33)-H(33) 0.93 0(1)#1-Co-N(1)#1 94.06(14) O(1)#1-Co-N(1)178.4(2) N(1)#1-Co-N(1) 87.58(5)87.5(2) N(1)#1-Co-N(2) 92.3(2) N(1B)-Co-N(2) 92.3(2) O(1)#1-Co-N(3) 89.3(2) N(1)#1-Co-N(3) 90.74(14) N(1B)-Co-N(3) 90.74(14) N(2)-Co-N(3) 175.8(3) N(1B)#1-N(1)-C(4) 107.767(5) N(1B)#1-N(1)-C(4B)#1 121.179(8) C(4)-N(1)-C(4B)#1 26.576(11) C(3)-N(1)-Co 126.8(4) C(4B)#1-N(1)-Co 104.54(3) N(1)#1-N(1B)-C(4)#1 72.232(5) N(1)#l-N(1B)-C(4B) 58.820(8)

Here we show that plasma kallikrein (PKal) mediates a plasminogen (Plg) cascade in adipocyte differentiation. Ecotin, an inhibitor of serine proteases, inhibits cell-shape change, adipocyte-specific gene expression, and lipid accumulation during adipogenesis in culture. Deficiency of Plg, but not of urokinase or tissue-type plasminogen activator, suppresses adipogenesis during differentiation of 3T3-L1 cells and mammary-gland involution. PKal, which is inhibited by ecotin, is required for adipose conversion, Plg activation and 3T3-L1 differentiation. Human plasma lacking PKal does not support differentiation of 3T3-L1 cells. PKal is therefore a physiological regulator that acts in the Plg cascade during adipogenesis. We propose that the Plg cascade fosters adipocyte differentiation by degradation of the fibronectin-rich preadipocyte stromal matrix.

Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-␣ antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (Kd ‫؍‬ 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and͞or three CDRs for each V H and VL domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-␣. These beneficial mutations in both VH and VL were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500-and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15-to 30-fold improvement in in vitro TNF-␣ neutralization in an L929 bioassay. Thus, this LTM͞combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins.look-through mutagenesis ͉ maturation ͉ mutagenesis ͉ TNF-␣

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