Proteolytic degradation of extracellular matrix (ECM) components during tissue remodeling plays a pivotal role in normal and pathological processes including wound healing, inflammation, tumor invasion, and metastasis. Proteolytic enzymes in tumors may activate or release growth factors from the ECM or act directly on the ECM itself, thereby facilitating angiogenesis or tumor cell migration. Fibroblast activation protein (FAP) is a cell surface antigen of reactive tumor stromal fibroblasts found in epithelial cancers and in granulation tissue during wound healing. It is absent from most normal adult human tissues. FAP is conserved throughout chordate evolution, with homologues in mouse and Xenopus laevis, whose expression correlates with tissue remodeling events. Using recombinant and purified natural FAP, we show that FAP has both dipeptidyl peptidase activity and a collagenolytic activity capable of degrading gelatin and type I collagen; by sequence, FAP belongs to the serine protease family rather than the matrix metalloprotease family. Mutation of the putative catalytic serine residue of FAP to alanine abolishes both enzymatic activities. Consistent with its in vivo expression pattern determined by immunohistochemistry, FAP enzyme activity was detected by an immunocapture assay in human cancerous tissues but not in matched normal tissues. This study demonstrates that FAP is present as an active cell surface-bound collagenase in epithelial tumor stroma and opens up investigation into physiological substrates of its novel, tumorassociated dipeptidyl peptidase activity.

We have generated a monoclonal antibody (mAb), 9EG7, against mouse endothelial cells that blocks adhesion of lymphocytes to endothelial cells. Sequencing offour tryptic peptides ofthe purified antigen revealed its identity with the integrin chain (3. The only Pu integrin that is known to mediate cell-cell adhesion is a4fi . This is not the integrin that is functionally dermed by the mAb 9EG7 on endothelial cells. First Antibodies. The following mAbs against mouse integrin chains were used: PS/2 (24) (anti-a4), R1-2 (16) (anti-a4), GoH3 (25) (anti-a6), and EA-1 (26, 27) (anti-a6). Other mAbs were K20 (28) (anti-human integrin chain (31) and (anti-mouse VCAM-1). Rabbit antibodies against a peptide corresponding to the C-terminal domain of the chicken integrin chain (31 were obtained from Richard 0. Hynes (MIT).The mAb 9EG7 (IgG2a) was produced by immunizing rats with TME cells in phosphate-buffered saline (PBS), using Alu-Gel-S (Serva) as adjuvant. Hybridoma production was done essentially as described (30) except that the mouse myeloma Sp2/0 was used for the fusion. Hybridoma supernatants were screened in cell-surface ELISA assays on TME Abbreviations: CAM, cell adhesion molecule; mAb, monoclonal antibody; PMA, phorbol 12-myristate 13-acetate.lTo whom reprint requests should be addressed. 9051

E-SELECTIN is an inducible cell-adhesion molecule on endothelial cells, which mediates the binding of neutrophils and functions as a Ca(2+)-dependent lectin. We have recently identified a 150K glycoprotein as the major ligand for E-selectin on myeloid cells, using a recombinant antibody-like form of mouse E-selectin as an affinity probe. Here we report the isolation of a mouse complementary DNA for this E-selectin ligand (ESL-1). The predicted amino-acid sequence of ESL-1 is 94% identical (over 1,078 amino acids) to the recently identified chicken cysteine-rich fibroblast growth-factor receptor, except for a unique 70-amino-acid aminoterminal domain of mature ESL-1. Fucosylation of ESL-1 is imperative for affinity isolation with E-selectin-IgG. A fucosylated, recombinant antibody-like form of ESL-1, but not of L-selectin, supports adhesion of E-selectin-transfected Chinese hamster ovary cells. Antibodies against ESL-1 block the binding of mouse myeloid cells to E-selectin. ESL-1, with a structure essentially identical to that of a receptor, thus functions as a cell adhesion ligand of E-selectin.

The cytoplasmic region of the Ca2+-dependent cell-adhesion molecule (CAM) uvomorulin associates with distinct cytoplasmic proteins with molecular masses of 102, 88, and 80 kDa termed a, (3, and ycatenin, respectively. This complex formation links uvomorulin to the actin filament network, which seems to be of primary importance for its cell-adhesion properties. We show here that antibodies against a catenin also immunoprecipitate complexes that contain human N-cadherin, mouse P-cadherin, chicken A-CAM (adherens junction-specific CAM; also called N-cadherin) or Xenopus U-cadherin, demonstrating that a catenin is complexed with other cadherins. In immunofluoresence tests, a catenin is colocalized with cadherins at the plasma membrane. However, in cadherin-negative Ltk-cells, a catenin is found uniformly distributed in the cytoplasm, suggesting some additional biological function(s). Expression of uvomorulin in these cells results in a concentration of a catenin at membrane areas ofcell contacts. We also have cloned and sequenced murine a catenin.The deduced amino acid sequence reveals a signfMcant homology to vinculin. Our results suggest the possibility of a new vinculin-related protein family involved in the cytoplasmic anchorage of cell-cell and cell-substrate adhesion molecules.The cadherin gene family of Ca2l-dependent cell adhesion molecules (CAM) was originally composed of a rather limited number of transmembrane glycoproteins of which the best studied examples were uvomorulin/E-cadherin, liver CAM (L-CAM), N-cadherin, and P-cadherin (for a review, see refs. 1 and 2). Each member was found to regulate cell adhesion of particular cell types, and this was thought to be fundamental for the organization of multicellular organisms. More recently new members of this family have been described including M-cadherin on mouse myoblasts (3), E/P-, U-, and XB-cadherin in early Xenopus development (4-6), and a new subgroup of more distantly related desmosomal glycoproteins (7-9).It has been shown that the cytoplasmic region of uvomorulin associates with defined proteins of 102, 88, and 80 kDa termed a, f3, and y catenin, respectively (10). The MATERIALS AND METHODSCell Lines. Mouse fibroblasts Ltk-, human HeLa, chicken fibroblasts CEF38, and their respective transfectants expressing mouse uvomorulin, Li-i, H1-3, and C1-4 (10, 15) were used as well as embryonal carcinoma cells F9, PCC4, and PAS5E. Porcine kidney LLC-PK7 and Xenopus A6 cells were gifts from H. Hoschutzky (Freiburg, F.R.G.) and D. Wedlich (Berlin), respectively. The A6 cells were grown in Leibovitz L-15 medium containing 8% (vol/vol) fetal calf serum (FCS) at 240C. All other cells were cultured in Dulbecco's modified Eagle's medium containing 10%o FCS at 370C in an atmosphere containing 10%6 Co2. For the generation of F9 tumors, about 1 x 107 cells were injected subcutaneously in 129/SV mice, and solid tumors were removed 12-15 days later and stored at -80TC.Puriication of a Catenin. Ten grams of solid F9 tumor was homogenized in 50 ml of No...

Abstract. E-and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca2+-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150-kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160-kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca 2+-dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide:N-glycosidase F inhibited cell binding to P-and E-select.in.In addition, glyeoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E-and P-selcctin in a Cae+-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospeciflc ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E-and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N-linked carbohydrates and is common for both endothelial selectins.

Endosialin, the antigen identified with monoclonal antibody FB5, is a highly restricted 165-kDa cell surface glycoprotein expressed by tumor blood vessel endothelium in a broad range of human cancers but not detected in blood vessels or other cell types in many normal tissues. Functional analysis of endosialin has been hampered by a lack of information about its molecular structure. In this study, we describe the purification and partial amino acid sequencing of endosialin, leading to the cloning of a full-length cDNA with an open reading frame of 2274 base pairs. The endosialin cDNA encodes a type I membrane protein of 757 amino acids with a predicted molecular mass of 80.9 kDa. The sequence matches with an expressed sequence tag of unknown function in public data bases, named TEM1, which was independently linked to tumor endothelium by serial analysis of gene expression profiling. Bioinformatic evaluation classifies endosialin as a C-type lectin-like protein, composed of a signal leader peptide, five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats), followed by a mucinlike region, a transmembrane segment, and a short cytoplasmic tail. Carbohydrate analysis shows that the endosialin core protein carries abundantly sialylated, O-linked oligosaccharides and is sensitive to O-sialoglycoprotein endopeptidase, placing it in the group of sialomucin-like molecules. The N-terminal 360 amino acids of endosialin show homology to thrombomodulin, a receptor involved in regulating blood coagulation, and to complement receptor C1qRp. This structural kinship may indicate a function for endosialin as a tumor endothelial receptor for as yet unknown ligands, a notion now amenable to molecular investigation.The endosialin antigen was identified in a survey of normal and neoplastic human tissues conducted at the Ludwig Institute for Cancer Research in pursuit of new targets for antibodybased cancer therapies (37). The hallmark of monoclonal antibody (mAb) 1 FB5, the probe used to discover endosialin (1), is its distinctive pattern of reactivity with human tissues. Thus, in a detailed study of biopsy and surgical specimens representing diverse cancer types, FB5 immunostaining was found primarily in tumor blood vessels and not in malignant tumor cells. Significantly, the antigen was not observed in all cancer samples examined, and even in cancers showing FB5-immunoreactive endothelial cells, the antigen was frequently detected with a heterogeneous pattern in the tumor vascular bed. Such a mixed pattern might be expected for a molecule involved in the reorganization of blood vessels in tissues such as cancers, in which areas of stable blood supply and histology are juxtaposed to regions of necrosis, hypoxia, excessive growth, tissue invasion, and remodeling. The normal tissues examined were unreactive with mAb FB5, including the blood vessel endothelium present in the respective organs. The expression of the FB5 antigen by cultured normal...

L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin–Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin–Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin–Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca2+, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin–Ig fusion protein was not observed. In agreement with the biochemical data, the anti–Lselectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin–Ig but not on P-selectin–Ig. No such inhibitory effect was seen with the anti–mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin–binding carbohydrate moieties are attached to different protein scaffolds in different species.

Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy.