Development of indole/indazole-aminopyrimidines as inhibitors of c-Jun N-terminal kinase (JNK): optimization for JNK potency and physicochemical properties.

A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design was used to optimize the series resulting in a significant potency improvement. The best compound (17) has IC50 of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other kinases with good physicochemical and pharmacokinetic properties.

Discovery of a novel series of 4-quinolone JNK inhibitors.

A novel series of highly selective JNK inhibitors based on the 4-quinolone scaffold was designed and synthesized. Structure based drug design was utilized to guide the compound design as well as improvements in the physicochemical properties of the series. Compound (13c) has an IC(50) of 62/170 nM for JNK1/2, excellent kinase selectivity and impressive efficacy in a rodent asthma model.

Development of an Improved Epstein-Barr Virus (EBV) Neutralizing Antibody Assay to Facilitate Development of a Prophylactic gp350-Subunit EBV Vaccine.

No licensed vaccine is available for prevention of EBV-associated diseases, and robust, high-throughput bioanalytical assays are needed to evaluate immunogenicity of gp350 subunit-based candidate EBV vaccines. Here we have developed an improved EBV-GFP based neutralization assay for such a vaccine's pre-clinical and clinical validation to measure EBV specific neutralizing antibodies in human donors. The supplementation of guinea pig complement of our previously published high-throughput EBV-GFP fluorescent focus (FFA)-based neutralization assay allowed the detection of complement-dependent neutralizing antibodies using a panel of heat-inactivated healthy human sera. Anti-gp350 antibody titers, which were evaluated using a previously optimized anti-gp350 IgG ELISA assay, were moderately correlated to the FFA-based neutralization titers. Overall, this improved high-throughput neutralization assay is capable of characterizing the serologic neutralizing antibody response to natural EBV infection, with applications in evaluating EBV antibody status in epidemiologic studies and immunogenicity of candidate gp350-subunit EBV vaccines in clinical studies.

Selective and non-selective metalloproteinase inhibitors reduce IL-1-induced cartilage degradation and loss of mechanical properties.

Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulated with proinflammatory cytokines such as interleukin-1 (IL-1). Aggrecanases and matrix metalloproteinases (MMPs) are thought to be principal downstream effectors of cytokine-induced matrix catabolism, and aggrecanase- or MMP-selective inhibitors reduce or block matrix destruction in several model systems. The objective of this study was to use metalloproteinase inhibitors to perturb IL-1-induced matrix catabolism in bovine cartilage explants and examine their effects on changes in tissue compression and shear properties. Explanted tissue was stimulated with IL-1 for up to 24 days in the absence or presence of inhibitors that were aggrecanase-selective, MMP-selective, or non-selective. Analysis of conditioned media and explant digests revealed that aggrecanase-mediated aggrecanolysis was delayed to varying extents with all inhibitor treatments, but that aggrecan release persisted. Collagen degradation was abrogated by MMP- and non-selective inhibitors and reduced by the aggrecanase inhibitor. The inhibitors delayed but did not reduce loss of the equilibrium compression modulus, whereas the losses of dynamic compression and shear moduli were delayed and reduced. The data suggest that non-metalloproteinase mechanisms participate in IL-1-induced matrix degradation and loss of tissue material properties.

Development of a High-Throughput Respiratory Syncytial Virus Fluorescent Focus-Based Microneutralization Assay.

Neutralizing antibodies specific for respiratory syncytial virus (RSV) represent a major protective mechanism against RSV infection, as demonstrated by the efficacy of the immune-prophylactic monoclonal antibody palivizumab in preventing RSV-associated lower respiratory tract infections in premature infants. Accordingly, the RSV neutralization assay has become a key functional method to assess the neutralizing activity of serum antibodies in preclinical animal models, epidemiology studies, and clinical trials. In this study, we qualified a 24-h, fluorescent focus-based microneutralization (RSVA FFA-MN) method that requires no medium exchange or pre- or postinfection processing to detect green fluorescent protein-expressing RSV strain A2 (RSVA-GFP)-infected cells, using a high-content imaging system for automated image acquisition and focus enumeration. The RSVA FFA-MN method was shown to be sensitive, with a limit of detection (LOD) and limit of quantitation (LOQ) of 1:10, or 3.32 log2; linear over a range of 4.27 to 9.65 log2 50% inhibitory concentration (IC50); and precise, with intra- and interassay coefficients of variation of <21%. This precision allowed the choice of a statistically justified 3-fold-rise seroresponse cutoff criterion. The repeatability and robustness of this method were demonstrated by including a pooled human serum sample in every assay as a positive control (PC). Over 3 years of testing between two laboratories, this PC generated data falling within 2.5 standard deviations of the mean 98.7% of the time (n = 1,720). This high-throughput and reliable RSV microneutralization assay has proven useful for testing sera from preclinical vaccine candidate evaluation studies, epidemiology studies, and both pediatric and adult vaccine clinical trials.

Development of a robust, higher throughput green fluorescent protein (GFP)-based Epstein-Barr Virus (EBV) micro-neutralization assay.

The goal of most prophylactic vaccines is to elicit robust and effective neutralizing antibodies against the human pathogen target. The titer of neutralizing antibodies to Epstein-Barr Virus (EBV) is a useful biomarker for evaluating EBV vaccines. Here, the development and optimization of a 96-well micro-neutralization fluorescent imaging assay (FIA) using an EBV virus-encoding green fluorescent protein (GFP) to infect adherent EBV recipient cells is reported. The conditions were optimized for generating reproducible EBV-GFP virus, for maintaining viral infectivity for months, and for efficient viral infection of recipient cell culture. The utility of the EBV-GFP FIA neutralization assay was demonstrated in a mouse study of an investigational adjuvanted EBV gp350 subunit vaccine. This assay confirmed the generation of high titers of anti-EBV-neutralizing antibodies which correlated well with the established Raji cell-based flow cytometry-based EBV neutralization assay, as well as with anti-gp350 IgG titers. In naturally infected EBV+ human serum samples, a good correlation between anti-gp350 IgG ELISA titer and EBV-GFP FIA neutralization antibody titer was also observed. Taken together, these results demonstrate the establishment of a scalable high throughput EBV-GFP FIA micro-neutralization assay suitable to measure humoral EBV vaccine response in a large-scale human trial.

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus.

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized "gold standard" panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the "gold standard" diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.

Differential gene expression profile of human tonsil high endothelial cells: implications for lymphocyte trafficking.

Lymphocyte recirculation is dependent on the interactions of adhesion and signaling molecules expressed on lymphocytes and their partners on high endothelial cells (HEC). Many of the events in this process have yet to be molecularly characterized. To identify novel HEC-specific proteins with potential function in the recruitment cascade, we sequenced a normalized human tonsil HEC cDNA library (generated from an inflamed tonsil) from which lymphocyte and human umbilical vein endothelial cell cDNAs had been subtracted. One-thousand forty-nine sequences were analyzed. All but three mapped to known cDNAs or genomic DNAs. The two most abundant transcripts encoded alpha2-macroglobulin and hevin. The next-abundant transcripts encoded several other protease inhibitors, making this protein class the most prominent in HEC. Several endothelial-specific transcripts were also identified, including those encoding E-selectin, vascular cell adhesion molecule-1, vascular endothelial-junctional adhesion molecule, and platelet-endothelial cell adhesion molecule-1. The library contains a great diversity of transcripts, and studies of the encoded proteins will provide further insight into the complex biology of these specialized endothelial cells.

Variations in aggrecan structure modulate its susceptibility to aggrecanases.

Proteoglycan aggregates and purified aggrecan from adult and fetal bovine cartilage and adult and neonatal human cartilage were subjected to in vitro degradation by recombinant aggrecanase-1 and aggrecanase-2. The ability of the aggrecanases to cleave within the aggrecan IGD (interglobular domain) and CS2 domain (chondroitin sulphate-rich domain 2) was monitored by SDS/PAGE and immunoblotting. Aggrecanase-2 showed a similar ability to cleave within the IGD of adult and immature aggrecan, whereas aggrecanase-1 was less efficient in cleavage in the IGD of immature aggrecan, for both the bovine and the human substrates. Both aggrecanases showed a similar ability to cleave within the CS2 domain of bovine aggrecan irrespective of age, but showed a much lower ability to cleave within the CS2 domain of human aggrecan. Equivalent results were obtained whether aggrecan was present in isolation or as part of proteoglycan aggregates. When proteoglycan aggregates were used, neither aggrecanase was able to cleave link protein. Thus, for aggrecan cleavage by aggrecanases, variations in cleavage efficiency exist with respect to the species and age of the animal from which the aggrecan is derived and the type of aggrecanase being used.

Identification of the critical attribute(s) of EBV gp350 antigen required for elicitation of a neutralizing antibody response in vivo.

Vaccine prophylaxis with EBV glycoprotein 350 (gp350) subunit plus adjuvant has been demonstrated clinically to protect individuals against infectious mononucleosis (IM), but the specifications of the antigen required to elicit this protection has remained largely theoretical. Previous studies have shown that antibodies to gp350 comprise the principle component of EBV-neutralizing sera. Further, a murine monoclonal antibody against gp350 (clone 72A1) is able to prevent infection by the virus both in vitro and in vivo. In the present study, we identify the 72A1 epitope on recombinant gp350 antigen as the site required for binding to CD21 on human B cells. We also identify the need for conformational-dependence of the antigen to generate EBV-neutralizing antibodies in vivo. Further, we have characterized the glycosylation status and antigenicity profiles of both native and denatured CHO-produced soluble gp350 as well as non-glycosylated protein produced in Escherichia coli. Collectively our in vitro and in vivo data demonstrate the requirement for a conformationally accessible 72A1 epitope on gp350 to elicit EBV-neutralizing responses, and establish this as a critical attribute of this vaccine antigen. These data provide direction for commercial vaccine development, as the absence of this epitope on either E. coli-expressed or denatured gp350, may limit production and purification options for the antigen.

Enhanced immunogenicity of a respiratory syncytial virus (RSV) F subunit vaccine formulated with the adjuvant GLA-SE in cynomolgus macaques.

Respiratory syncytial virus (RSV) causes significant disease in elderly adults, but an effective vaccine is not yet available. We have previously reported that vaccines consisting of engineered respiratory syncytial virus soluble fusion protein (RSV sF) adjuvanted with glucopyranosyl lipid A (GLA) in an oil-in-water emulsion (stable emulsion [SE]) induce RSV F-specific T and B cell responses in mice and rats that protect from viral challenge. Here, we evaluated the immunogenicity of GLA-SE adjuvanted RSV sF vs unadjuvanted RSV sF vaccines in cynomolgus macaques (Macaca fascicularis). RSV F-specific IgG, RSV neutralizing antibodies, and RSV F-specific T cell IFNγ ELISPOT responses induced by GLA-SE adjuvanted RSV sF peaked at week 6 at significantly higher levels than achieved by unadjuvanted RSV sF and remained detectable through week 24, demonstrating response longevity. Two weeks after a week 24 booster immunization, humoral and cellular responses reached levels similar to those seen at the earlier peak response. Importantly, the GLA-SE adjuvanted RSV sF vaccine induced cross-neutralizing antibodies to other RSV A and B strains as well as F-specific IgA and IgG memory B cells. GLA-SE adjuvanted RSV sF was also demonstrated to drive a Th1-biased response characterized by more IFNγ than IL-4. This study indicates that a GLA-SE adjuvanted RSV sF vaccine induces robust humoral and Th1-biased cellular immunity in non-human primates and may benefit human populations at risk for RSV disease.

Adults 65 years old and older have reduced numbers of functional memory T cells to respiratory syncytial virus fusion protein.

Respiratory syncytial virus (RSV) infects elderly (≥65 years) adults, causing medically attended illness and hospitalizations. While RSV neutralizing antibody levels correlate inversely with RSV-associated hospitalization in the elderly, the role of RSV-specific T cells in preventing disease in the elderly remains unclear. We examined RSV-specific humoral, mucosal, and cellular immune profiles in healthy elderly (65 to 85 years) and young (20 to 30 years) adults. RSV neutralization antibody titers in the elderly (10.5 ± 2.2 log(2)) and young (10.5 ± 2.1 log(2)) were similar. In contrast, levels of RSV F protein-specific gamma interferon (IFN-γ)-producing T cells were lower in elderly (180 ± 80 spot-forming cells [SFC]/10(6) peripheral blood mononuclear cells [PBMC]) than in young adults (1,250 ± 420 SFC/10(6) PBMC). Higher levels of interleukin-13 (IL-13; 3,000 ± 1,000 pg/ml) in cultured PBMC supernatants and lower frequency of RSV F-specific CD107a(+) CD8(+) T cells (3.0% ± 1.6% versus 5.0% ± 1.6%) were measured in PBMC from elderly than young adults. These results suggest that deficient RSV F-specific T cell responses contribute to susceptibility to severe RSV disease in elderly adults.

Implication of respiratory syncytial virus (RSV) F transgene sequence heterogeneity observed in Phase 1 evaluation of MEDI-534, a live attenuated parainfluenza type 3 vectored RSV vaccine.

MEDI-534 is the first live vectored RSV vaccine candidate to be evaluated in seronegative children. It consists of the bovine parainfluenza virus type 3 (PIV3) genome with substituted human PIV3 F and HN glycoproteins engineered to express RSV F protein. A Phase 1 study of 49 healthy RSV and PIV3 seronegative children 6 to <24 months of age demonstrated an acceptable safety profile at the following doses: 10(4), 10(5) and 10(6)TCID50. After 3 doses of MEDI-534 at 10(6)TCID50, administered at 0, 2 and 4 month intervals, 100% of subjects seroresponded to PIV3, whereas only 50% seroresponded to RSV. To investigate the discordance in seroresponse rates, the RSV F transgene and its flanking non-coding nucleotides were sequenced from shed virus recovered from the nasal washes of 24 MEDI-534-vaccinated children. Eleven out of 24 samples contained no nucleotide changes in the analyzed region. The other 13 samples contained mixtures of variant subpopulations. Fifty-five percent exhibited changes in the transcription termination poly A gene sequences of the upstream bPIV3N gene while 21% had variant subpopulations in the RSV F open reading frame that resulted in pre-mature stop codons. Both types of changes are expected to reduce RSV F expression. Evaluation of the administered vaccine by dual immunofluorescence staining showed ~2.5% variants with low or no RSV F expression while single nucleotide primer extension detected ~1% variation at nucleotide 2045 that resulted in a pre-mature translational termination at codon 85. An association between shedding of variants and lower RSV F serological response was observed but it was not possible to establish a definitive clinical significance due to the small number of subjects in this study.

Translational sciences approach to RSV vaccine development.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and the elderly. Despite its relatively low degree of antigenic variation, it causes frequent reinfection throughout life. Clinical manifestations of RSV disease and the immune response to infection differ in infants and the elderly, suggesting that vaccines designed to protect these two populations may require different attributes. Here, the authors describe the translational approach of utilizing data from epidemiology studies performed in these populations, the use of RSV diagnostics in clinical practice, lessons learned from previous vaccine clinical trials and the success of palivizumab in prevention of RSV disease in premature and high-risk infants to aid the development of safe and effective RSV vaccines.

Safety and immunogenicity of a live attenuated RSV vaccine in healthy RSV-seronegative children 5 to 24 months of age.

UNLABELLED: Despite substantial morbidity associated with respiratory syncytial virus (RSV) infection, there is no licensed vaccine. MEDI-559 is a live attenuated intranasal vaccine candidate being developed for prevention of lower respiratory illness due to RSV in young children. This randomized, placebo-controlled study evaluated safety of MEDI-559 in healthy, RSV-seronegative children. MEDI-559 or placebo was administered on 3 occasions, 2 months apart. Primary safety was based on solicited symptoms (SSs) and adverse events (AEs) collected for 28 days after each dose. Nasal wash samples were collected 3 times after each dose (days 7-10, 12-18, 28-34) and at sick visits. Serum was collected for measuring antibody immune responses to RSV prior to first vaccination and 28 days post final dose. Long-term safety was monitored for 365 days from first dose. SSs were mild and frequent (MEDI-559 84%; placebo 91%); most common SSs were runny/stuffy nose, cough, and irritability/fussiness. AEs occurred in 67% MEDI-559 and 57% placebo recipients: most common AE was upper respiratory tract infection (MEDI-559 35%; placebo 23%). Higher incidence of medically attended lower respiratory illness within 28 days after dosing occurred in the MEDI-559 arm compared to placebo (none associated with vaccine virus shedding). There was no evidence of enhanced RSV disease. Vaccine virus was detected only in MEDI-559 recipients; shedding occurred in 56%subjects, primarily post dose 1. A functional immune response was observed in 59% and 9% MEDI-559 and placebo recipients, respectively, by an RSV microneutralization assay. Vaccine take, assessed by proportion that shed vaccine-type virus or had a seroresponse against RSV, was seen in 95% MEDI-559 subjects. MEDI-559 is therefore biologically active and immunogenic in this seronegative pediatric population. Although the frequency of SSs and AEs was not considered clinically significant, the increase in medically attended lower respiratory illnesses in the vaccine group warrants expanded safety studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT00767416.

Aggrecanolysis and in vitro matrix degradation in the immature bovine meniscus: mechanisms and functional implications.

INTRODUCTION: Little is known about endogenous or cytokine-stimulated aggrecan catabolism in the meniscal fibrocartilage of the knee. The objectives of this study were to characterize the structure, distribution, and processing of aggrecan in menisci from immature bovines, and to identify mechanisms of extracellular matrix degradation that lead to changes in the mechanical properties of meniscal fibrocartilage. METHODS: Aggrecanase activity in the native immature bovine meniscus was examined by immunolocalization of the aggrecan NITEGE neoepitope. To investigate mechanisms of cytokine-induced aggrecan catabolism in this tissue, explants were treated with interleukin-1alpha (IL-1) in the absence or presence of selective or broad spectrum metalloproteinase inhibitors. The sulfated glycosaminoglycan (sGAG) and collagen contents of explants and culture media were quantified by biochemical methods, and aggrecan catabolism was examined by Western analysis of aggrecan fragments. The mechanical properties of explants were determined by dynamic compression and shear tests. RESULTS: The aggrecanase-generated NITEGE neoepitope was preferentially localized in the middle and outer regions of freshly isolated immature bovine menisci, where sGAG density was lowest and blood vessels were present. In vitro treatment of explants with IL-1 triggered the accumulation of NITEGE in the inner and middle regions. Middle region explants stimulated with IL-1 exhibited substantial decreases in sGAG content, collagen content, and mechanical properties. A broad spectrum metalloproteinase inhibitor significantly reduced sGAG loss, abrogated collagen degradation, and preserved tissue mechanical properties. In contrast, an inhibitor selective for ADAMTS-4 and ADAMTS-5 was least effective at blocking IL-1-induced matrix catabolism and loss of mechanical properties. CONCLUSIONS: Aggrecanase-mediated aggrecanolysis, typical of degenerative articular cartilage, may play a physiologic role in the development of the immature bovine meniscus. IL-1-induced release of sGAG and loss of mechanical properties can be ascribed primarily to the activity of MMPs or aggrecanases other than ADAMTS-4 and ADAMTS-5. These results may have implications for the clinical management of osteoarthritis.

Multimodal signaling by the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) promotes neurite extension.

Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains associate with hyaluronan and various secreted proteins to form a complex of extracellular matrix (ECM) that inhibits neural plasticity in the central nervous system (CNS). Chondroitinase treatment depletes PGs of their CS side chains and enhances neurite extension. Increasing evidence from in vivo models indicates that proteolytic cleavage of the PG core protein by members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of glutamyl-endopeptidases also promotes neural plasticity. The purpose of this study was to determine whether proteolytic action of the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of primary rat neurons with ADAMTS4 cDNA induced longer neurites, whether the neurons were grown on a monolayer of astrocytes that secrete inhibitory PGs or on laminin/poly-L-lysine substrate alone. Similar results were found when neurons were transfected with a construct encoding a proteolytically inactive, point mutant of ADAMTS4. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures also enhanced neurite extension in a dose-dependent manner, an effect demonstrated to be dependent on the activation of MAP ERK1/2 kinase. These results suggest that ADAMTS4 enhances neurite outgrowth via a mechanism that does not require proteolysis but is dependent on activation of the MAP kinase cascade. Thus a model to illustrate multimodal ADAMTS activity would entail proteolysis of CS-bearing PGs to create a loosened matrix environment more favorable for neurite outgrowth, and enhanced neurite outgrowth directly stimulated by ADAMTS signaling at the cell surface.

Kinetic characterization of human JNK2alpha2 reaction mechanism using substrate competitive inhibitors.

Jun N-terminal kinase (JNK) is a stress activated serine/threonine protein kinase that phosphorylates numerous cellular protein substrates including the transcription factors c-Jun and ATF2. In this study, we defined the kinetic mechanism for the active form of JNK2alpha2. Double reciprocal plots of initial rates versus concentrations of substrate revealed the sequential nature of the JNK2alpha2 catalyzed ATF2 phosphorylation. Dead-end JNK inhibitors were then used to differentiate ordered and random kinetic mechanisms for the reaction. A peptide inhibitor containing the homology JNK docking sequence for substrate recognition, derived from amino acid residues 153-163 of JNK-interacting protein 1 (JIP-1), inhibited JNK activity via competition with ATF2. This peptide functioned as a noncompetitive inhibitor against ATP. In contrast, the anthrapyrazolone compound, SP600125, exhibited competitive inhibition for ATP and noncompetitive inhibition against ATF2. Furthermore, binding of one substrate had no significant effect on the affinity for the other substrate. The data in this study are consistent with a kinetic mechanism for activated JNK2alpha2 in which (1) substrate binding is primarily due to the distal contacts in the JNK2alpha2 docking groove that allow the delivery of the substrate phosphorylation sequence into the catalytic center, (2) there is minimal allosteric communication between the protein-substrate docking site and the ATP binding site in the catalytic center for activated JNK2alpha2, and (3) the reaction proceeds via a random sequential mechanism.

ADAMTS4 (aggrecanase-1) activation on the cell surface involves C-terminal cleavage by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on syndecan-1.

C-terminal truncation of ADAMTS-4 from the p68 form to the p53 form is required for activation of its capacity to cleave the Glu(373)-Ala(374) interglobular domain bond of aggrecan. In transfected human chondrosarcoma cells, this process is not autoproteolytic because the same products form with an inactive mutant of ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin-like motif 4) and truncation is completely blocked by tissue inhibitor of metalloproteinase-1. Instead, activation can be mediated by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase (MT4-MMP, MMP-17) because co-transfection with the active form of MT4-MMP markedly enhanced activation, whereas an inactive mutant of MT4-MMP was ineffective. Treatment of co-transfected cells with phosphatidylinositol-specific phospholipase C liberated the complex of MT4-MMP and p68 ADAMTS4 from the cell membrane, but the p53 ADAMTS4 remained associated. Specific glycosaminoglycan lyase digestions, followed by product analyses using fluorescence-assisted carbohydrate electrophoresis and immunoprecipitation experiments, showed that the p53 form is associated with syndecan-1 through both chondroitin sulfate and heparan sulfate. We conclude that ADAMTS-4 activation in this cell system involves the coordinated activity of both glycosylphosphatidyl inositol-anchored MT4-MMP and the proteoglycan form of syndecan-1 on the cell surface.