Shib D. Banerjee

Molecular characterization of a novel intracellular hyaluronan-binding protein.

Hyaluronan has well defined functions in extracellular matrices and at the surface of cells. However, several studies have now shown that significant pools of hyaluronan are also present intracellularly, but its function therein is unknown. One avenue of investigation that may assist in defining the function of intracellular hyaluronan is to identify intracellular hyaluronan-binding proteins. In previous studies we identified CDC37, a cell cycle regulatory protein, using a monoclonal antibody that recognizes a novel group of hyaluronan-binding proteins. In this study, we have identified a second hyaluronan-binding protein with this antibody and characterized its properties. This protein, which we have termed IHABP4, was also found to be an intracellular and a specific hyaluronan-binding protein, containing several hyaluronan-binding motifs: (R/K)X 7(R/K) (where R/K denotes arginine or lysine and X denotes non-acidic amino acids). Furthermore, we have determined the gene organization ofIHABP4 and cloned cDNAs for the chick, mouse, and human homologs. Comparison of the deduced chick, mouse, and human protein sequences showed that the hyaluronan-binding motifs, (R/K)X 7(R/K), in these sequences are conserved; both chick and mouse IHABP4 were shown directly to bind hyaluronan. Biochemical fractionation and immunofluorescent localization of epitope-tagged IHABP4 indicated that it is mainly present in the cytoplasm. These data support the possibility that intracellular hyaluronan and its binding proteins may play important roles in cell behavior.

Monoclonal antibody to chick embryo hyaluronan-binding protein: Changes in distribution of binding protein during early brain development

A monoclonal antibody, MAb IVd4, that recognizes hyaluronan-binding protein (HABP) from chick embryo brain has been produced and characterized. By immunoblotting, MAb IVd4 was shown to recognize three proteins in chick embryo brain of molecular weight 93, 90, and 69 kDa; this interaction was inhibited by addition of hyaluronan hexasaccharides. Overlay of transblots with [3H]hyaluronan showed binding to proteins of similar molecular weight. MAb IVd4 blocked binding of [3H]hyaluronan to brain HABP and to simian virus-transformed 3T3 cells, indicating a possible relationship with the 85-kDa hyaluronan receptor of these cells. The distribution of HABP during early brain development was analyzed by immunohistochemistry. Immunoreactivity was uniform in newly formed neuroectoderm but became more concentrated in the roof of the brain during the second day of embryonic development. As the neuroectoderm becomes layered, the HABP was increasingly restricted to the forming plexiform layer, an area enriched in neural cell processes. Immunoreactivity was greatly enhanced by pretreatment of tissue with hyaluronidase, presumably due to removal of hyaluronan bound to the HABP, and was abolished on treatment with hyaluronan hexasaccharide, presumably due to inhibition of HABP-antibody interaction. These results suggest that a hyaluronan receptor is involved in early cellular events in brain development.

Hyaluronan-Cell Interactions in Limb Development

Hyaluronan is a ubiquitous component of the extracellular matrices in which cells migrate and proliferate during embryonic development (reviewed in Toole, 1981). Its physical and chemical properties contribute to an extracellular milieu which is important both to the structural integrity of embryonic tissues and to the morphogenetic processes that take place within them. One way in which hyaluronan participates in tissue structure arises from its ability to form meshworks that exert osmotic pressure (Comper and Laurent, 1978; Meyer, 1983). The resultant swelling pressure within the tissue can lead to separation of cellular or fibrous structures or deformation of the tissue, possibly facilitating cell and tissue movements (Toole, 1981; Morris-Wiman and Brinkley, 1990). In addition, some embryonic cells exhibit large, hyaluronan-dependent, pericellular coats (Knudson and Toole, 1985) that may influence cell-cell adhesion (Underhill and Toole, 1981; Knudson, 1990a), cell-substratum adhesion (Barnhart et al., 1979), cell proliferation (Brecht et al., 1986), migration (Turley et al., 1985; Schor et al., 1989) or differentiation (Kujawa et al., 1986).

Detection of a specific pattern of hyaluronan oligosaccharides and their binding proteins in human ovarian tumour

Tumour cells generate hyaluronan (HA) oligomers (O‐HA) by an autocrine mechanism to regulate their own behaviour through receptor interaction, necessitating analysis of HA sizes and its receptor expression in tumour progression. In this study for the first time, we identified specific size of HA in malignant ovarian tumour compared to benign tumour tissue. Therefore, we prepared the identified HA probes and conducted multiplex and monoplex ligand blot analysis and Immunohistochemistry to identify their receptor expression and distribution. Although, HA recognized CD44 as principle receptors despite of size, multiplex analysis showed multiple receptor expression with distribution at the tumour cell surface. Furthermore, the HA 6‐mer (major O‐HA of ovarian tumour) pull down of tumour tissue proteins showed 120 kDa protein along with CD44 with over expression in the malignant tumour. Upon depletion of CD44 protein HA 6‐mer showed a major 120 kDa protein with distribution at nuclear membrane, suggesting that this protein may play an important role in ovarian tumour progression. In summary, ovarian tumour cells of different grade showed heterogeneity in generation of HA oligomers and their interaction with specific receptors. Therefore, simultaneous analysis of O‐HA and their receptors expression could serve as a prognostic indicator during tumorigenesis. Copyright

IDENTIFICATION OF A NOVEL INTRACELLULAR HYALURONAN-BINDING PROTEIN, IHABP4

ABSTRACT The functions of hyaluronan in extracellular matrices and at the surface of cells have been extensively studied. However, it is now clear that significant pools of hyaluronan are also present intracellularly and, in an attempt to define the function of intracellular hyaluronan, we have sought to identify intracellular hyaluronan-binding proteins. In previous studies we identified CDC37, a cell cycle regulatory protein, using a monoclonal antibody that recognizes a novel group of hyaluronan-binding proteins. In this study, we have identified a second hyaluronan-binding protein with this antibody, and characterized its properties. This protein, which we term IHABP4, is an intracellular and specific hyaluronan-binding protein containing several hyaluronan-binding motifs, namely B(X7)B (where B denotes basic and X denotes non-acidic amino acids). Comparison of the deduced chick, mouse and human protein sequences indicates that the hyaluronan-binding motifs in these sequences are conserved. By biochemical fractionation and immunofluorescent localization of epitope-tagged IHABP4 we have shown that it is mainly present in the cytoplasm. We also confirmed the intracellular localization of hyaluronan in actively cycling cells. These data support the possibility that intracellular hyaluronan and its binding proteins may play important roles in cell behavior.