Fat1 cadherin provides a novel minimal residual disease marker in acute lymphoblastic leukemia.

Measurement of minimal residual disease (MRD) maintains an important role in the clinical management of acute lymphoblastic leukemia (ALL). Recently, we identified Fat1 cadherin as a unique and independent prognostic factor for relapse-free and overall survival in pediatric pre-B-ALL. Here, we analyzed Fat1 mRNA for its potential as a novel marker of MRD in cases of pre-B- and T-ALL. Analyses of microarray data from 125 matched diagnosis/relapse samples across three independent datasets indicate that Fat1 mRNA is detectable in an average of 31.3% of diagnosed pre-B-ALL, of which 67.5% of cases remained positive at relapse. Furthermore, some 20% of cases with undetectable levels of Fat1 mRNA at diagnosis became positive upon relapse. T-ALL cases were 83.3% positive for Fat1 expression at diagnosis with 77.7% remaining positive at relapse. Towards proof of concept, we developed a quantitative polymerase chain reaction assay and demonstrate detection of Fat1 mRNA in leukemic cells mixed with normal peripheral blood cells at a sensitivity of 1 in 10 000 to 100 000 cells. Fat1 may therefore provide a new marker of MRD for patients with ALL lacking known genomic aberrations or within a multiplex approach to MRD detection.

Dual processing of FAT1 cadherin protein by human melanoma cells generates distinct protein products.

The giant cadherin FAT1 is one of four vertebrate orthologues of the Drosophila tumor suppressor fat. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development. Homozygous deletions in oral cancer suggest that FAT1 may play a tumor suppressor role, although overexpression of FAT1 has been reported in some other cancers. Here we show using Northern blotting that human melanoma cell lines variably but universally express FAT1 and less commonly FAT2, FAT3, and FAT4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that, like Drosophila FAT, human FAT1 is cleaved into a non-covalent heterodimer before achieving cell surface expression. The use of inhibitors also established that such cleavage requires the proprotein convertase furin. However, in melanoma cells, the non-cleaved proform of FAT1 is also expressed at the cell surface together with the furin-cleaved heterodimer. Moreover, furin-independent processing generates a potentially functional proteolytic product in melanoma cells, a persistent 65-kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. In vitro localization studies of FAT1 showed that melanoma cells display high levels of cytosolic FAT1 protein, whereas keratinocytes, despite comparable FAT1 expression levels, exhibited mainly cell-cell junctional staining. Such differences in protein distribution appear to reconcile with the different protein products generated by dual FAT1 processing. We suggest that the uncleaved FAT1 could promote altered signaling, and the novel products of alternate processing provide a dominant negative function in melanoma.

Palmitoylation of CD36/FAT regulates the rate of its post-transcriptional processing in the endoplasmic reticulum.

CD36/FAT is a transmembrane glycoprotein that functions in the cellular uptake of long-chain fatty acids and also as a scavenger receptor. As such it plays an important role in lipid homeostasis and, pathophysiologically, in the progression of type 2 diabetes and atherosclerosis. CD36 expression is tightly regulated at the levels of both transcription and translation. Here we show that its expression and location are also regulated post-translationally, by palmitoylation. Although palmitoylation of CD36 was not required for receptor maturation and cell surface expression, inhibition of palmitoylation either pharmacologically with cerulenin or by mutation of the relevant cysteines delayed processing at the ER and trafficking through the secretory pathway. The absence of palmitoylation also reduced the half life of the CD36 protein. Additionally, the CD36 palmitoylation mutant did not incorporate efficiently into lipid rafts, a site known to be required for its function of fatty acid uptake, and this reduced the efficiency of uptake of oxidized low density lipoprotein. These findings provide an added level of sophistication where translocation of CD36 to the plasma membrane may be physiologically regulated by palmitoylation.

CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis.

Atherosclerotic plaques result from the excessive deposition of cholesterol esters derived from lipoproteins and lipoprotein fragments. Tissue macrophage within the intimal space of major arterial vessels have been shown to play an important role in this process. We demonstrate in a transfection system using two human cell lines that the macrophage scavenger receptor CD36 selectively elicited lipid uptake from Cu(2+)-oxidized high density lipoprotein (HDL) but not from native HDL or low density lipoprotein (LDL). The uptake of oxHDL displayed morphological and biochemical similarities with the CD36-dependent uptake of oxidized LDL. CD36-mediated uptake of oxidized HDL by macrophage may therefore contribute to atheroma formation.

Shed gangliosides provide detergent-independent evidence for type-3 glycosynapses.

Membrane microdomains, or rafts, at the plasma membrane have been invoked to explain many cellular processes. Protein-protein interactions within such microdomains including, for example, the tetraspanin web are reported to provide a scaffold for signal transduction. However, the nature of such protein-protein interactions is not fully elucidated. Hakomori [S.I. Hakomori, The glycosynapse, Proc. Natl. Acad. Sci. USA 99 (2002) 225-232] has advanced the concept that glycosphingolipids, particularly gangliosides, provide the intermediary link between transmembrane receptors and signal transducers and has redefined membrane rafts as Type-1, -2 or -3 glycosynapses. Here, using simple immunofluorescent analysis of the ganglioside complexes naturally released from cellular microprocesses (termed "footprints") we show that the ganglioside can determine the nature of protein-protein associations. Specifically, we demonstrate that CD36 and the tetraspanin CD151, both of which interact with beta1 integrins, associate together only in the presence of the gangliosides GD2/GD3. These results substantiate the glycosynapse hypothesis and suggest that the nature of the tetraspanin web may be determined by gangliosides.

Novel immunoblotting monoclonal antibodies against human and rat CD36/fat used to identify an isoform of CD36 in rat muscle.

CD36, a surface membrane glycoprotein, functions as a class B scavenger receptor that binds to several distinct ligands. Included among these is oxidized low-density lipoprotein (Ox-LDL), a major trigger of atherosclerotic lesions, and the levels of CD36 activity and Ox-LDL uptake may have an impact on coronary artery disease. In addition, recent studies in rodents have shown that CD36, also known as FAT, controls the levels of free fatty acids and triglycerides in plasma, and is an important regulator of the metabolic pathways involved in insulin resistance. Despite the importance of measuring CD36 expression in different tissues there is a paucity of good immunoblotting antibodies, particularly for rodent tissue. Here, using GST-fusion proteins incorporating the cysteine cluster encoded by exons VIII, IX, and X of the CD36 gene as immunogen, we have generated a panel of monoclonal antibodies that are excellent blotting reagents for human and rat CD36. With these reagents we were able to visualize an additional, faster migrating CD36 band in rat muscle, likely representing a minor splice variant of CD36 (CD36var.1) hitherto seen only in the human HEL cell line.

Cloning and expression of the large zebrafish protocadherin gene, Fat.

The cadherin superfamily members play an important role in mediating cell-cell contact and adhesion (Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455). A distinct subfamily, neither belonging to the classical or protocadherins includes Fat, the largest member of the cadherin super-family. Fat was originally identified in Drosophila. Subsequently, orthologues of Fat have been described in man (Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q., Beverley, P. C., Owen, M. J., 1995. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30, 207-223), rat (Ponassi, M., Jacques, T. S., Ciani, L., ffrench, C. C., 1999. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80, 207-212) and mouse (Cox, B., Hadjantonakis, A. K., Collins, J. E., Magee, A. I., 2000. Cloning and expression throughout mouse development of mfat1, a homologue of the Drosophila tumour suppressor gene fat [In Process Citation]. Dev. Dyn. 217, 233-240). In Drosophila, Fat has been shown to play an important role in both planar cell polarity and cell boundary formation during development. In this study we describe the characterization of zebrafish Fat, the first non-mammalian, vertebrate Fat homologue to be identified. The Fat protein has 64% amino acid identity and 80% similarity to human FAT and an identical domain structure to other vertebrate Fat proteins. During embryogenesis fat mRNA is expressed in the developing brain, specialised epithelial surfaces the notochord, ears, eyes and digestive tract, a pattern similar but distinct to that found in mammals.