Results Melanoma Transfectants Expressing CD 44 H Bind HA More Effectively Than the Same Cells Expressing CD 44

The hyaluronan (HA)-binding function (lectin function) of the leukocyte homing receptor, CD44, is tightly regulated. Herein we address possible mechanisms that regulate CD44 isoform-specific H A binding. Binding studies with melanoma transfectants expressing CD44H, CD44E, or with soluble immunoglobulin fusions of CD44H and CD44E (CD44H-Rg, CD44ERg) showed that although both CD44 isoforms can bind HA, CD44H binds H A more efficiently than CD44E. Using CD44-Rg fusion proteins we show that the variably spliced exons in CD44E, V8-V10, specifically reduce the lectin function of CD44, while replacement of V8-V10 by an ICAM-1 immunoglobulin domain restores binding to a level comparable to that of CD44H. Conversely, CD44 bound HA very weakly when exons V8-V10 were replaced with a CD34 mucin domain, which is heavily modified by O-linked glycans. Production of CD44E-Rg or incubation of CD44Eexpressing transfectants in the presence of an O-linked glycosylation inhibitor restored HA binding to CD44HRg and to cell surface CD44H levels, respectively. We conclude that differential splicing provides a regulatory mechanism for CD44 lectin function and that this effect is due in part to O-linked carbohydrate moieties which are added to the Ser/Thr rich regions encoded by the variably spliced CD44 exons. Alternative splicing resuiting in changes in protein glycosylation provide a novel mechanism for the regulation of lectin activity. C D44 represents a heterogeneous group of cell surface and secreted proteins generated by alternate splicing of a single gene (47). All CD44 isoforms contain at the amino terminus an HA-binding domain which is composed of two clusters of positively charged amino acids (44, 55). Protein heterogeneity arises predominantly from the variable splicing of exons encoding extracellular domains located between the invariant hyaluronan(HA) 1binding domains and exons encoding the membrane proximal extracellular domain (13,23, 33). Alternative splicing of exons encoding the cytoplasmic domain of CD44 has also been reported (15, 47). To date at least 18 different alternatively spliced CD44 isoforms have been reported. Cells can express more than one CD44 isoform at a time, however, in some cell types one CD44 isoform is preferentially expressed. Leukocytes predominantly express CD44H (50), an isoform whose extracellular domain contains no variably spliced extracellular domain exons, while epitheAddress all correspondence to Dr. Kelly L. Bennett, BMS-PRI, 3005 First Avenue, Seattle, W A 98121. Tel. : (206) 727-3569. Fax: (206) 727-3602. 1. Abbreviat ion used in this paper. HA, hyaluronan; MC, melanoma cells; RT-PCR, reverse transcriptase-polymerase chain reaction. lial cells express many alternatively spliced isoforms (8, 17, 30, 51). The CD44 isoforms expressed by a given cell can also change in response to differentiation and/or activation (1, 28) suggesting that the contribution of CD44 to the function of a given cell is not always constant. These observations suggest that these different CD44 molecules are endowed with distinct and possibly multiple functions. Indeed, in vitro evidence supporting this hypothesis has been reported. CD44 is the cell surface receptor for HA and there is evidence that the binding capacities of the various isoforms differ (22, 27, 44, 51). In addition, it has been shown that the CD44 isoforms whose extracellular domain contains variably spliced exon V3 can be modified with heparan sulfate and bind to a subset of heparin-binding growth factors (6, 22). Other CD44 functions have not been so clearly assigned to particular isoforms, nonetheless, CD44 is a widely distributed multifunctional glycoprotein. Among its many physiological functions, CD44 has been implicated in leukocyte homing (25), leukocyte activation (20, 43, 46), extracellular matrix binding (2, 9, 24, 54), and cell adhesion and migration (49, 53). Recent experiments have shown that abnormal expression of CD44 by tumor cells can enhance their ability to grow and/or metastasize in vivo suggesting that abnormal expres© The Rockefeller University Press, 0021-9525/95/12/1623/11 $2.00 The Journal of Cell Biology, Volume 131, Number 6, Part 1, December 1995 1623-1633 1623 on A uust 3, 2017 jcb.rress. org D ow nladed fom sion and/or regulation of CD44 function may play a role in some human cancers (5, 17, 51). A number of different experimental results lead to the conclusion that CD44 functions as a lectin by binding HA. These include the demonstration that the binding of H A to cells expressing CD44 can be blocked by anti-CD44 mAbs (32, 35, 41), the finding that a soluble immunoglobulin fusion of CD44H can bind H A in a cell-free system (44), and the demonstration that a protein recognized by mAb K3 which binds to the hamster HA-receptor (52) is the hamster homologue of CD44H (2). These results were further supported by observations that anti-CD44 mAbs could precipitate [3H]HA-binding activity from murine and human cell lines, block the binding of labeled-HA to the receptor and inhibit the HA-dependent aggregation of CD44-expressing cells (9, 16). In some cell lines the lectin function of CD44 is regulated (32, 40). Lesley et al. (32) showed that a number of cell lines which expressed high levels of CD44 bind H A only after activation with phorbol esters. This observation has been confirmed using additional cell lines (21, 31), peripheral blood lymphocytes (14), and transfected cell lines (35, 38), clearly establishing that in some cell types CD44 lectin function is tightly regulated. Additional studies indicate that high affinity binding of CD44 for soluble H A requires an intact cytoplasmic domain (35, 38), CD44 aggregation on the cell surface (34), protein synthesis (42), and cytoplasmic domain phosphorylation (45). Also, there is evidence that different CD44 isoforms have different HAbinding capacities suggesting that alternative splicing plays a role in the regulation of the lectin function of CD44 (22, 51). Here we report on the results of experiments designed to examine the role of alternative splicing in the regulation of CD44 lectin function. Using CD44-negative cell lines which were transfected to express either CD44H or CD44E, an isoform with an extracellular domain containing variably spliced exons V8-V10, we found that cells expressing CD44H bound H A more effectively than cells expressing CD44E. This difference was also seen with soluble immunoglobulin fusion of these two CD44 isoforms. Using soluble forms of CD44 which contain peptide sequences encoded by one or more of the variably spliced exons found in CD44E or with a polypeptide fragment obtained from ICAM-1, we show that inclusion of the CD44 variably spliced exons specifically reduces the ability of CD44E to bind HA. We also present the results of experiments which suggest that the reduced HA-binding capacity of CD44E is due in part to the presence of O-linked carbohydrates which were added to the Ser/Thr rich regions found in the variably spliced exons of CD44E (38, 44, 51). Materials and Methods Cell Culture and Fusion Protein Expression CD44 stable transfected human melanoma cell lines, melanoma cells (MC), have been previously described (53). MC44H, MC44E, and MC44E-trunc express wild-type CD44H, wild-type CD44E, and a CD44E deletion mutant which is missing exon E15 and E16 (the junction between V10 and E17 is NVNRSWLI), respectively. COS cells were purchased from Amer. Type Culture Collection (Rockville, MD), grown in DMEM/ 10% FBS, and used for transient expression of fusion proteins as previously described (2). To generate O-linked deglycosylated CD44E-Rg, and CD44H-Rg, 1 mM phenyl-c~-N-acetylgalactosaminide (phenyl-c~-GalNAc) (Sigma Immunochemicals, St. Louis, MO) was added to DMEM during the protein production phase of the transfection, and to the melanoma transfectants for 16 h before testing HA binding.