Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.

Lymph node stromal cells (LNSCs) closely regulate immunity and self-tolerance, yet key aspects of their biology remain poorly elucidated. Here, comparative transcriptomic analyses of mouse LNSC subsets demonstrated the expression of important immune mediators, growth factors and previously unknown structural components. Pairwise analyses of ligands and cognate receptors across hematopoietic and stromal subsets suggested a complex web of crosstalk. Fibroblastic reticular cells (FRCs) showed enrichment for higher expression of genes relevant to cytokine signaling, relative to their expression in skin and thymic fibroblasts. LNSCs from inflamed lymph nodes upregulated expression of genes encoding chemokines and molecules involved in the acute-phase response and the antigen-processing and antigen-presentation machinery. Poorly studied podoplanin (gp38)-negative CD31(-) LNSCs showed similarities to FRCs but lacked expression of interleukin 7 (IL-7) and were identified as myofibroblastic pericytes that expressed integrin α(7). Together our data comprehensively describe the transcriptional characteristics of LNSC subsets.

Antioxidant functions of DHHC3 suppress anti-cancer drug activities.

Ablation of protein acyltransferase DHHC3 selectively enhanced the anti-cancer cell activities of several chemotherapeutic agents, but not kinase inhibitors. To understand why this occurs, we used comparative mass spectrometry-based palmitoyl-proteomic analysis of breast and prostate cancer cell lines, ± DHHC3 ablation, to obtain the first comprehensive lists of candidate protein substrates palmitoylated by DHHC3. Putative substrates included 22-28 antioxidant/redox-regulatory proteins, thus predicting that DHHC3 should have antioxidant functions. Consistent with this, DHHC3 ablation elevated oxidative stress. Furthermore, DHHC3 ablation, together with chemotherapeutic drug treatment, (a) elevated oxidative stress, with a greater than additive effect, and (b) enhanced the anti-growth effects of the chemotherapeutic agents. These results suggest that DHHC3 ablation enhances chemotherapeutic drug potency by disabling the antioxidant protections that contribute to drug resistance. Affirming this concept, DHHC3 ablation synergized with another anti-cancer drug, PARP inhibitor PJ-34, to decrease cell proliferation and increase oxidative stress. Hence, DHHC3 targeting can be a useful strategy for selectively enhancing potency of oxidative stress-inducing anti-cancer drugs. Also, comprehensive identification of DHHC3 substrates provides insight into other DHHC3 functions, relevant to in vivo tumor growth modulation.

Integrin-independent support of cancer drug resistance by tetraspanin CD151.

Tetraspanin protein CD151 has typically been studied as binding partner and functional regulator of laminin-binding integrins. However, we show here that CD151 supports anti-cancer drug resistance independent of integrins. CD151 ablation sensitized multiple tumor cell types to several anti-cancer drugs (e.g., gefitinib and camptothecin), thus increasing apoptosis, as seen using cleaved caspase-3, cleaved PARP (poly (ADP-ribose) polymerase), annexin V, and propidium iodide staining assays. Drug sensitization due to CD151 ablation is integrin-independent, because, (1) effects occurred in cells when integrins were unengaged with ligand, (2) integrin ablation (α3 and α6 subunits) did not mimic effects of CD151 ablation, (3) the CD151QRD mutant, with diminished integrin association, and CD151WT (unmutated CD151) similarly reconstituted drug protection, and (4) treatment with anti-cancer drugs selectively upregulated intracellular nonintegrin-associated CD151 (NIA-CD151), consistent with its role in drug resistance. Together, these results suggest that upregulated CD151 expression may support not only typical integrin-dependent functions, but also integrin-independent survival of circulating (and possibly metastatic) cancer cells during anti-cancer drug therapy.

Tetraspanin TSPAN12 regulates tumor growth and metastasis and inhibits ß-catenin degradation.

Ablation of tetraspanin protein TSPAN12 from human MDA-MB-231 cells significantly decreased primary tumor xenograft growth, while increasing tumor apoptosis. Furthermore, TSPAN12 removal markedly enhanced tumor-endothelial interactions and increased metastasis to mouse lungs. TSPAN12 removal from human MDA-MB-231 cells also caused diminished association between FZD4 (a key canonical Wnt pathway receptor) and its co-receptor LRP5. The result likely explains substantially enhanced proteosomal degradation of ß-catenin, a key effecter of canonical Wnt signaling. Consistent with disrupted canonical Wnt signaling, TSPAN12 ablation altered expression of LRP5, Naked 1 and 2, DVL2, DVL3, Axin 1, and GSKß3 proteins. TSPAN12 ablation also altered expression of several genes regulated by ß-catenin (e.g. CCNA1, CCNE2, WISP1, ID4, SFN, ME1) that may help to explain altered tumor growth and metastasis. In conclusion, these results provide the first evidence for TSPAN12 playing a role in supporting primary tumor growth and suppressing metastasis. TSPAN12 appears to function by stabilizing FZD4-LRP5 association, in support of canonical Wnt-pathway signaling, leading to enhanced ß-catenin expression and function.

CD151 restricts the α6 integrin diffusion mode.

Laminin-binding integrins (α3ß1, α6ß1, α6ß4, α7ß1) are almost always expressed together with tetraspanin CD151. In every coexpressing cell analyzed to date, CD151 makes a fundamental contribution to integrin-dependent motility, invasion, morphology, adhesion and/or signaling. However, there has been minimal mechanistic insight into how CD151 affects integrin functions. In MDA-MB-231 mammary cells, tetraspanin CD151 knockdown impairs α6 integrin clustering and functions without decreasing α6 integrin expression or activation. Furthermore, CD151 knockdown minimally affects the magnitude of α6 integrin diffusion, as measured using single particle tracking. Instead, CD151 knockdown has a novel and unexpected dysregulating effect on the mode of α6 integrin diffusion. In control cells α6 integrin shows mostly random-confined diffusion (RCD) and some directed motion (DMO). In sharp contrast, in CD151-knockdown cells α6 integrin shows mostly DMO. In control cells α6 diffusion mode is sensitive to actin disruption, talin knockdown and phorbol ester stimulation. By contrast, CD151 knockdown cell α6 integrin is sensitive to actin disruption but desensitized to talin knockdown or phorbol ester stimulation, indicating dysregulation. Both phorbol ester and EGF stimulate cell spreading and promote α6 RCD in control cells. By contrast, CD151-ablated cells retain EGF effects but lose phorbol-ester-stimulated spreading and α6 RCD. For α6 integrins, physical association with CD151 promotes α6 RCD, in support of α6-mediated cable formation and adhesion. By comparison, for integrins not associated with CD151 (e.g. αv integrins), CD151 affects neither diffusion mode nor αv function. Hence, CD151 support of α6 RCD is specific and functionally relevant, and probably underlies diverse CD151 functions in skin, kidney and cancer cells.

The C-terminal tail of tetraspanin protein CD9 contributes to its function and molecular organization.

Tetraspanin protein CD9 supports sperm-egg fusion, and regulates cell adhesion, motility, metastasis, proliferation and signaling. The large extracellular loop and transmembrane domains of CD9 engage in functionally important interactions with partner proteins. However, neither functional nor biochemical roles have been shown for the CD9 C-terminal tail, despite it being highly conserved throughout vertebrate species. To gain new insight into the CD9 tail, three C-terminal amino acids (Glu-Met-Val) were replaced with residues corresponding to C-terminal amino acids from tetraspanin protein CD82 (Pro-Lys-Tyr). Wild-type and mutant CD9 were then stably expressed in MOLT-4, K562, U937, RD and HT1080 cells. Whereas wild-type CD9 inhibited cell adhesion and spreading on fibronectin, mutant CD9 did not. Wild-type CD9 also promoted homotypic cell-cell aggregation and microvilli formation, whereas mutant CD9 did not. Protein interactions of wild-type and mutant CD9 were compared quantitatively using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with liquid-chromatography-tandem mass spectrometry (LC-MS/MS) technology. SILAC results showed that, despite wild-type and mutant CD9 having identical expression levels, mutant CD9 and its major transmembrane interacting partners were recovered in substantially reduced amounts from 1% Brij 96 lysates. Immunoprecipitation experiments confirmed that mutant CD9 recovery was decreased in Brij 96, but not in more stringent Triton X-100 detergent. Additionally, compared with wild-type CD9 complexes, mutant CD9 complexes were larger and more oligomerized in Brij 96 detergent, consistent with decreased Brij 96 solubility, perhaps due to more membrane domains packing more tightly together. In conclusion, multiple CD9 functions depend on its C-terminal tail, which affects the molecular organization of CD9 complexes, as manifested by their altered solubilization in Brij 96 and organization on the cell surface.

Diminished metastasis in tetraspanin CD151-knockout mice.

Tetraspanin protein CD151 on tumor cells supports invasion and metastasis. In the present study, we show that host animal CD151 also plays a critical role. CD151-null mice showed markedly diminished experimental lung metastasis after injection of Lewis lung carcinoma or B16F10 melanoma cells. Diminished tumor cell residence in the lungs was evident 6-24 hours after injection. Consistent with an endothelial cell deficiency, isolated CD151-null mouse lung endothelial cells showed diminished support for B16F10 adhesion and transendothelial migration, diminished B16F10-induced permeability, and diminished B16F10 adhesion to extracellular matrix deposited by CD151-null mouse lung endothelial cells. However, CD151 deletion did not affect the size of metastatic foci or subcutaneous primary B16F10 tumors, tumor aggregation, tumor clearance from the blood, or tumor-induced immune cell activation and recruitment. Therefore, the effects of host CD151 on metastasis do not involve altered local tumor growth or immune surveillance. VEGF-induced endothelial cell signaling through Src and Akt was diminished in CD151-null endothelial cells. However, deficient signaling was not accompanied by reduced endothelial permeability either in vitro (monolayer permeability assay) or in vivo (VEGF-stimulated Miles assay). In summary, diminished metastasis in CD151-null host animals may be due to impaired tumor-endothelial interactions, with underlying defects in mouse lung endothelial cell extracellular matrix production.

Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin α6ß4.

The laminin-binding integrin α6ß4 plays key roles in both normal epithelial and endothelial cells and during tumor cell progression, metastasis, and angiogenesis. Previous cysteine mutagenesis studies have suggested that palmitoylation of α6ß4 protein supports a few integrin-dependent functions and molecular associations. Here we took another approach and obtained strikingly different results. We used overexpression and RNAi knockdown in multiple cell types to identify protein acyl transferase DHHC3 as the enzyme responsible for integrin ß4 and α6 palmitoylation. Ablation of DHHC3 markedly diminished integrin-dependent cellular cable formation on Matrigel, integrin signaling through Src, and ß4 phosphorylation on key diagnostic amino acids (S1356 and 1424). However, unexpectedly, and in sharp contrast to prior α6ß4 mutagenesis results, knockdown of DHHC3 accelerated the degradation of α6ß4, likely due to an increase in endosomal exposure to cathepsin D. When proteolytic degradation was inhibited (by Pepstatin A), rescued α6ß4 accumulated intracellularly, but was unable to reach the cell surface. DHHC3 ablation effects were strongly selective for α6ß4. Cell-surface levels of ~10 other proteins (including α3ß1) were not diminished, and the appearance of hundreds of other palmitoylated proteins was not altered. Results obtained here demonstrate a new substrate for the DHHC3 enzyme and provide novel opportunities for modulating α6ß4 expression, distribution, and function.

Tetraspanin CD151 protects against pulmonary fibrosis by maintaining epithelial integrity.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disorder of unknown etiology with few treatment options. Although tetraspanins are involved in various diseases, their roles in fibrosis have not been determined. OBJECTIVES: To investigate the role of tetraspanin CD151 in pulmonary fibrosis. METHODS: CD151 knockout (KO) mice were studied by histological, biochemical, and physiological analyses and compared with wild-type mice and CD9 KO mice. Further mechanistic analyses were performed in vitro, in vivo, and on samples from patients with IPF. MEASUREMENTS AND MAIN RESULTS: A microarray study identified an enrichment of genes involved in connective tissue disorders in the lungs of CD151 KO mice, but not in CD9 KO mice. Consistent with this, CD151 KO mice spontaneously exhibited age-related pulmonary fibrosis. Deletion of CD151 did not affect pulmonary fibroblast functions but instead degraded epithelial integrity via attenuated adhesion strength on the basement membrane; CD151-deleted alveolar epithelial cells exhibited increased α-SMA expression with activation of p-Smad2, leading to fibrotic changes in the lungs. This loss of epithelial integrity in CD151 KO lungs was further exacerbated by intratracheal bleomycin exposure, resulting in severe fibrosis with increased mortality. We also observed decreased numbers of CD151-positive alveolar epithelial cells in patients with IPF. CONCLUSIONS: CD151 is essential for normal function of alveolar epithelial cells; loss of CD151 causes pulmonary fibrosis as a result of epithelial disintegrity. Given that CD151 may protect against fibrosis, this protein represents a novel target for the treatment of fibrotic diseases.

Antioxidant and Anticancer Functions of Protein Acyltransferase DHHC3.

Silencing of DHHC3, an acyltransferase enzyme in the DHHC family, extensively upregulates oxidative stress (OS). Substrates for DHHC3-mediated palmitoylation include several antioxidant proteins and many other redox regulatory proteins. This helps to explain why DHHC3 ablation upregulates OS. DHHC3 also plays a key role in cancer. DHHC3 ablation leads to diminished xenograft growth of multiple cancer cell types, along with diminished metastasis. Furthermore, DHHC3 protein is upregulated on malignant/metastatic cancer samples, and upregulated gene expression correlates with diminished patient survival in several human cancers. Decreased primary tumor growth due to DHHC3 ablation may be partly explained by an elevated OS → senescence → innate immune cell recruitment mechanism. Elevated OS due to DHHC3 ablation may also contribute to adaptive anticancer immunity and impair tumor metastasis. In addition, DHHC3 ablation disrupts antioxidant protection mechanisms, thus enhancing the efficacy of OS-inducing anticancer drugs. A major focus has thus far been on OS regulation by DHHC3. However, remaining to be studied are multiple DHHC3 substrates that may affect tumor behavior independent of OS. Nonetheless, the currently established properties of DHHC3 make it an attractive candidate for therapeutic targeting in situations in which antioxidant protections need to be downmodulated, and also in cancer.

Tetraspanin protein contributions to cancer.

Among the 33 human tetraspanin proteins, CD151, CD9 and Tspan12 play particularly important roles in cancer. Tetraspanin CD151, in partnership with integrins α6ß1 and α6ß4, modulates tumour cell growth, invasion, migration, metastasis, signalling and drug sensitivity. Tetraspanin CD9 has suppressor functions in multiple tumour cell types. Major CD9 partner proteins, such as EWI-2 and EWI-F, may modulate these tumour-suppressor functions. Tetraspanin Tspan12 mutations are linked to a human disease called familial exudative vitreoretinopathy. In addition, as a regulator of the metalloprotease ADAM10 (a disintegrin and metalloprotease 10) maturation and function, Tspan12 probably contributes to the pro-tumorigenic functions of ADAM10.

Targeting of tetraspanin proteins--potential benefits and strategies.

The tetraspanin transmembrane proteins have emerged as key players in malignancy, the immune system, during fertilization and infectious disease processes. Tetraspanins engage in a wide range of specific molecular interactions, occurring through the formation of tetraspanin-enriched microdomains (TEMs). TEMs therefore serve as a starting point for understanding how tetraspanins affect cell signalling, adhesion, morphology, motility, fusion and virus infection. An abundance of recent evidence suggests that targeting tetraspanins, for example, by monoclonal antibodies, soluble large-loop proteins or RNAi technology, should be therapeutically beneficial.

Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein.

Using mass spectrometry, we identified ADAM10 (a membrane-associated metalloproteinase) as a partner for TSPAN12, a tetraspanin protein. TSPAN12-ADAM10 interaction was confirmed by reciprocal coimmunoprecipitation in multiple tumor cell lines. TSPAN12, to a greater extent than other tetraspanins (CD81, CD151, CD9, and CD82), associated with ADAM10 but not with ADAM17. Overexpression of TSPAN12 enhanced ADAM10-dependent shedding of amyloid precursor protein (APP) in MCF7 (breast cancer) and SH-SY5Y (neuroblastoma) cell lines. Conversely, siRNA ablation of endogenous TSPAN12 markedly diminished APP proteolysis in both cell lines. Furthermore, TSPAN12 overexpression enhanced ADAM10 prodomain maturation, whereas TSPAN12 ablation diminished ADAM10 maturation. A palmitoylation-deficient TSPAN12 mutant failed to associate with ADAM10, inhibited ADAM10-dependent proteolysis of APP, and inhibited ADAM10 maturation, most likely by interfering with endogenous wild-type TSPAN12. In conclusion, TSPAN12 serves as a novel and robust partner for ADAM10 and promotes ADAM10 maturation, thereby facilitating ADAM10-dependent proteolysis of APP. This novel mode of regulating APP cleavage is of relevance to Alzheimer's disease therapy.

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5.

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.

An extracellular site on tetraspanin CD151 determines alpha 3 and alpha 6 integrin-dependent cellular morphology.

The alpha 3 beta 1 integrin shows strong, stoichiometric, direct lateral association with the tetraspanin CD151. As shown here, an extracellular CD151 site (QRD(194-196)) is required for strong (i.e., Triton X-100-resistant) alpha 3 beta 1 association and for maintenance of a key CD151 epitope (defined by monoclonal antibody TS151r) that is blocked upon alpha 3 integrin association. Strong CD151 association with integrin alpha 6 beta 1 also required the QRD(194-196) site and masked the TS151r epitope. For both alpha 3 and alpha 6 integrins, strong QRD/TS151r-dependent CD151 association occurred early in biosynthesis and involved alpha subunit precursor forms. In contrast, weaker associations of CD151 with itself, integrins, or other tetraspanins (Triton X-100-sensitive but Brij 96-resistant) were independent of the QRD/TS151r site, occurred late in biosynthesis, and involved mature integrin subunits. Presence of the CD151-QRD(194-196)-->INF mutant disrupted alpha 3 and alpha 6 integrin-dependent formation of a network of cellular cables by Cos7 or NIH3T3 cells on basement membrane Matrigel and markedly altered cell spreading. These results provide definitive evidence that strong lateral CD151-integrin association is functionally important, identify CD151 as a key player during alpha 3 and alpha 6 integrin-dependent matrix remodeling and cell spreading, and support a model of CD151 as a transmembrane linker between extracellular integrin domains and intracellular cytoskeleton/signaling molecules.

Palmitoylation supports assembly and function of integrin-tetraspanin complexes.

As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (alpha3, alpha6, and beta4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of beta4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient beta4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core alpha6beta4-CD151 complex formation was unaltered. There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading. Notably, beta4 palmitoylation neither increased localization into "light membrane" fractions of sucrose gradients nor decreased solubility in nonionic detergents-hence it does not promote lipid raft association. Instead, palmitoylation of beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.

Functional domains in tetraspanin proteins.

Exciting new findings have emerged about the structure, function and biochemistry of tetraspanin proteins. Five distinct tetraspanin regions have now been delineated linking structural features to specific functions. Within the large extracellular loop of tetraspanins, there is a variable region that mediates specific interactions with other proteins, as well as a more highly conserved region that has been suggested to mediate homodimerization. Within the transmembrane region, the four tetraspanin transmembrane domains are probable sites of both intra- and inter-molecular interactions that are crucial during biosynthesis and assembly of the network of tetraspanin-linked membrane proteins known as the 'tetraspanin web'. In the intracellular juxtamembrane region, palmitoylation of cysteine residues also contributes to tetraspanin web assembly, and the C-terminal cytoplasmic tail region could provide specific functional links to cytoskeletal or signaling proteins.

Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of beta-catenin in prostate cancer.

Fatty acid synthase (FASN), a key metabolic enzyme for liponeogenesis highly expressed in several human cancers, displays oncogenic properties such as resistance to apoptosis and induction of proliferation when overexpressed. To date, no mechanism has been identified to explain the oncogenicity of FASN in prostate cancer. We generated immortalized prostate epithelial cells (iPrECs) overexpressing FASN, and found that (14)C-acetate incorporation into palmitate synthesized de novo by FASN was significantly elevated in immunoprecipitated Wnt-1 when compared to isogenic cells not overexpressing FASN. Overexpression of FASN caused membranous and cytoplasmic beta-catenin protein accumulation and activation, whereas FASN knockdown by short-hairpin RNA resulted in a reduction in the extent of beta-catenin activation. Orthotopic transplantation of iPrECs overexpressing FASN in nude mice resulted in invasive tumors that overexpressed beta-catenin. A strong significant association between FASN and cytoplasmic (stabilized) beta-catenin immunostaining was found in 862 cases of human prostate cancer after computerized subtraction of the membranous beta-catenin signal (P<0.001, Spearman's rho=0.33). We propose that cytoplasmic stabilization of beta-catenin through palmitoylation of Wnt-1 and subsequent activation of the pathway is a potential mechanism of FASN oncogenicity in prostate cancer.

Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells: central role of CD81 in facilitating GPR56-Galpha q/11 association.

By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein-coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Galpha(q), Galpha(11), and Gbeta, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Galpha(q/11) complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Galpha(q/11) complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.

Links between CD147 function, glycosylation, and caveolin-1.

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-catalyzed, beta1,6-branched, polylactosamine-type sugars, which account for its excess size. Therefore, CD147, which is itself elevated on invasive tumor cells, may make a major contribution to the abundance of beta1,6-branched polylactosamine sugars that appear on invasive tumor cells. It was shown previously that caveolin-1 associates with CD147, thus inhibiting CD147 self-aggregation and MMP induction; now we show that caveolin-1 associates with LG-CD147 and restricts the biosynthetic conversion of LG-CD147 to HG-CD147. In addition, HG-CD147 (but not LG-CD147) was preferentially captured as a multimer after treatment of cells with a homobifunctional cross-linking agent and was exclusively recognized by monoclonal antibody AAA6, a reagent that selectively recognizes self-associated CD147 and inhibits CD147-mediated MMP induction. In conclusion, we have 1) determined the biochemical basis for the unusual size variation in CD147, 2) established that CD147 is a major carrier of beta1,6-branched polylactosamine sugars on tumor cells, and 3) determined that caveolin-1 can inhibit the conversion of LG-CD147 to HG-CD147. Because it is HG-CD147 that self-aggregates and stimulates MMP induction, we now have a mechanism to explain how caveolin-1 inhibits these processes. These results help explain the previously established tumor suppressor functions of caveolin-1.