Golgi localization and dynamics of hyaluronan binding protein 1 (HABP1/p32/C1QBP) during the cell cycle (original) (raw)
Related papers
2004
Hyaluronan (HA)-binding protein 1 (HABP1) is multifunctional in nature and exists as a trimer through coiled-coil interaction between α-helices at its N-and C-termini. To investigate the importance of trimeric assemblage and HA-binding ability of HABP1, we generated and overexpressed variants of HABP1 by truncating the α-helices at its termini. Subsequently, these variants were transiently expressed in COS-1 cells to examine the influence of these structural variations on normal cell morphology, as compared with those imparted by HABP1. Substantiating the centrality of coiled-coil interaction for maintaining the trimeric assembly of HABP1, we demonstrate that disruption of trimerization does not alter the affinity of variants towards its ligand HA. Transient expression of HABP1 altered the morphology of COS-1 cells by generating numerous cytoplasmic vacuoles along with disruption of the f-actin network. Interestingly, the truncated variants also imparted identical morphological changes. Characterization of the cytoplasmic vacuoles revealed that most of these vacuoles were autophagic in nature, resembling those generated under stress conditions. The identical morphological changes manifested in COS-1 cells on transient expression of HABP1 or its variants is attributed to their comparable HA-binding ability, which in concert with endogenous HABP1, may deplete the cellular HA pool. Such quenching of HA below a threshold level in the cellular milieu could generate a stress condition, manifested through cytoplasmic vacuoles and a disassembly of the f-actin network.
Molecular Characterization of a Novel Intracellular Hyaluronan-binding Protein
Journal of Biological Chemistry, 2000
Background. ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates. Methodology/Principal Findings. Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1) is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained. Conclusions/Significance. Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.
Hyaluronan in cytosol--Microinjection-based probing of its existence and suggested functions
Glycobiology, 2013
Hyaluronan (HA) is a large glycosaminoglycan produced by hyaluronan synthases (HAS), enzymes normally active at plasma membrane. While HA is delivered into the extracellular space, intracellular HA is also seen, mostly in vesicular structures, but there are also reports on its presence in the cytosol and specific locations and functions there. We probed the possibility of HA localization and functions in cytosol by microinjecting fluorescent HA binding complex (fHABC), HA fragments and hyaluronidase (HYAL) into cytosol. Microinjection of fHABC did not reveal HA-specific intracellular binding sites. Likewise, specific cytosolic binding sites for HA were not detected, as microinjected fluorescent HA composed of 4-8 monosaccharide units (HA4-HA8) were evenly distributed throughout the cells, including the nucleus, but excluded from membrane-bound organelles. The largest HA tested ( HA120 or 25 kDa) did not enter the nucleus, and HA10-HA28 were progressively excluded from parts of nuclei resembling nucleoli. In contrast, HA oligosaccharides endocytosed from medium remained in vesicular compartments. The activity of HA synthesis was estimated by measuring the HA coat on green fluorescent protein (GFP)-HAS3-transfected MCF-7 cells. Microinjection of HA4 reduced coat size at 4 h, but increased at 24 h after injection, while larger HA-oligosaccharides and HYAL had no influence. As a positive control, microinjection of glucose increased coat size. In summary, no evidence for the presence or function of HA in cytosol was obtained. Also, the synthesis of HA and the active site of HAS were not accessible to competition, binding and degradation by cytosolic effectors, while synthesis responded to increased substrate supply.
Molecular Reproduction and Development, 2008
Ovulation is a complex process of releasing a fertilizable oocyte and depends on the proper formation of an extracellular hyaluronan rich matrix by the cumulus oocyte complex (COC). The formation of a HA rich matrix is dependent on the synthesis and organization of HA in the presence of several biomolecules that mediate its crosslinking. To gain an insight into the follicular maturation and COC expansion, we have studied the expression of hyaluronan binding protein 1 (HABP1), which is known to interact specifically with hyaluronan. The level of HABP1 increased markedly during ovulation after gonadotropin stimulation, and the overexpression was seen in mural granulosa cells, expanding cumulus cells and follicular fluid. However, HABP1 could not be detected in the luteal cells of corpus luteum after ovulation. Such increased expression of HABP1 was observed both during in vivo and in vitro conditions of COC expansion. The level of HABP1 transcript was upregulated up to fivefold after COC expansion as compared to compact COC. Immunofluorescence analysis showed HABP1 to be localized in the cytoplasm and extracellular matrix, suggesting its role in ECM organization. The cultured expanded COC treated with hyaluronidase for different time periods showed the gradual dispersion of COC, which coincide with the loss of HABP1 from the matrix suggesting that HABP1 is bound to hyaluronan. These results indicate that HABP1 expressed in rat COCs during maturation may facilitate the formation of the HA matrix in the extracellular space around the oocyte with cumulus expansion during maturation. Mol. Reprod. Dev. 75: 429–438, 2008. © 2007 Wiley-Liss, Inc.
Hyaluronan-binding Proteins: Tying Up the Giant
Journal of Biological Chemistry, 2001
The ubiquitous glycosaminoglycan (GAG) 1 hyaluronan has diverse biological roles in vertebrates. These include acting as a vital structural component of connective tissues, the formation of loose hydrated matrices that allow cells to divide and migrate (e.g. during development), immune cell adhesion and activation, and a role in intracellular signaling (1-3). This wide range of activities may seem surprising for an unbranched polysaccharide comprised entirely of a repeating disaccharide, D-glucuronic acid(133)N-acetyl-D-glucosamine(134), which (unlike other GAGs) is neither attached to a protein core nor O-or N-sulfated. Such diversity results in fact from the large number of hyaluronan-binding proteins (often termed hyaladherins) that exhibit significant differences in their tissue expression, cellular localization, specificity, affinity, and regulation. Therefore, characterization of the molecular basis of hyaluronan recognition by proteins and how this is modulated in vivo is an important key to understanding the biology of this GAG. In this article, we review the structural organization of vertebrate hyaladherins and how this may contribute to their different biological activities.
Intracellular hyaluronan: Importance for cellular functions
Seminars in Cancer Biology, 2020
Hyaluronan-rich matrices are abundant in ECM and are involved in biological processes, such as cell growth and migration. Hyaluronan is synthesized by the hyaluronan synthase family of enzymes, HAS1, HAS2 and HAS3; the HAS1 and HAS3 genes give rise to different transcripts through alternative splicing, and the HAS2 gene to a non-coding RNA antisense transcript in addition to the protein-coding transcript. Biosynthesis of hyaluronan increases during inflammation and cancer and is regulated by cytokines and growth factors. In addition to extracellular hyaluronan-rich matrices, cytoplasmic and nuclear forms of hyaluronan have been detected in normal and pathological processes. Extra-and intra-cellular hyaluronan binds to hyaluronan binding proteins, such as CD44, RHAMM, CDC37 and USP17, affecting cellular behavior. Although neither the exact mechanisms by which hyaluronan is present in the intracellular compartments, nor its function at these sites are currently understood, there are evidence that intracellular hyaluronan has important regulatory roles during cell cycle, cell motility, RNA translation and splicing, and autophagy.
Increased hyaluronate synthesis is required for fibroblast detachment and mitosis
Biochemical Journal, 1986
Human-embryo fibroblasts were synchronized by means of colchicine and cytochalasin, and the production of hyaluronate was determined by [3H]glucosamine incorporation and ion-exchange chromatography. Cells arrested by colchicine synthesized small amounts of hyaluronate, whereas cells blocked by cytochalasin were stimulated in hyaluronate production. When the colchicine block was released, there was an increased synthesis of hyaluronate, which appeared first in the cellular fraction and was then shed into the culture medium. After release of the cytochalasin block, the hyaluronate production declined to that found with unsynchronized cells. A comparable increase of hyaluronate synthase activity was observed during mitosis. When hyaluronate synthesis was blocked by periodate-oxidized UDP-glucuronic acid, the cells were arrested in mitosis before rounding of cells. These results suggest that hyaluronate synthesis is required for detachment and rounding of cells during mitosis.
Journal of Biological Chemistry, 2012
Background: Hyaluronan (HA) levels regulate cell behavior, tumor invasion, and migration through interactions with hyaladherins. Results: Elevated expression of hyaluronan-binding protein 1 (HABP1) leads to enhanced HA synthesis, HA cable formation, and activation of cell survival pathways in HepG2 cells. Conclusion: Constitutively elevated expression of HABP1 leads to enhanced tumorigenic potential by HA-mediated pathways. Significance: HABP1 modulates cell survival through enhanced HA synthesis. Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.
The Journal of biological chemistry, 2015
In vertebrates, hyaluronan is produced in plasma membrane from cytosolic UDP-sugar substrates by HAS1-3 isoenzymes that transfer N-Acetyl glucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou et al. 2010 J. Biol. Chem. 285 23647-23654). Here we have systematically screened in live cells potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, an...