Effects of nerve growth factor-induced differentiation on the heparan sulfate of PC12 pheochromocytoma cells and comparison with developing brain

We have examined the size, charge, and sulfation pattern of heparan sulfate in the cell-soluble fraction, membranes, and culture medium of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF) and compared the structural features of PC12 cell heparan sulfate to that of rat brain at several stages of early postnatal development. Nitrous acid degradation studies revealed significant differences in the distribution of N-sulfate and N-acetyl groups in heparan sulfate present in the PC12 cell-soluble fraction, membranes, and medium and demonstrated that NGF treatment led to an increased proportion of N-sulfated segments in the cell-associated heparan sulfate, although no such change was seen in that released into the culture medium. There was very little change in the N-sulfation of brain heparan sulfate during the first 30 days after birth. In brain, most of the heparan sulfate glucosamine residues are N-sulfated and yield predominantly di- and tetrasaccharide nitrous acid degradation products, whereas PC12 cell heparan sulfate contains large blocks of N-acetylglucosamine residues. There was very little difference in the overall charge or size (approximately 15,000 Da) of heparan sulfate chains between the different PC12 cell fractions or brain, although NGF treatment led to a decrease in the proportion of less-charged chains in the PC12 cell membranes and a small increase in molecular size. Our studies therefore demonstrate the presence in PC12 cells of several pools of heparan sulfate having different structural properties, and that significant alterations in the charge, size, and sulfation pattern of PC12 cell heparan sulfate accompany NGF-induced differentiation and neurite outgrowth.     

Disulfide-bonded aggregates of heparan sulfate proteoglycans

Heparan sulfate proteoglycans have been isolated from Swiss mouse 3T3 cells by using two nondegradative techniques: extraction with 4 M guanidine or 2.5% 1-butanol. These proteoglycans were separated from copurifying chondroitin sulfate proteoglycans by using ion-exchange chromatography on DEAE-cellulose in the presence of 2 M urea. The purified heparan sulfate proteoglycans are substantially smaller, ca. Mr 20 000, than those isolated from these same cells with trypsin, ca. Mr 720 000 [Johnston, L.S., Keller, K. L., & Keller, J. M. (1979) Biochim. Biophys. Acta 583, 81-94]. However, all of the heparan sulfate proteoglycans extracted by these three methods contain similar glycosaminoglycan chains (Mr 7500) and are derived from the same pool of cell surface associated molecules. The trypsin-released heparan sulfate proteoglycan (ca. Mr 720 000) can be significantly reduced in size (ca. Mr 33 000) under strong denaturing conditions in the presence of the disulfide reducing agent dithiothreitol, which suggests that this form of the molecule is a disulfide-bonded aggregate. The heparan sulfate proteoglycan isolated from the medium also undergoes a significant size reduction in the presence of dithiothreitol, indicating that a similar aggregate is formed as part of the normal release of heparan sulfate proteoglycans into the medium. These results suggest that well-shielded disulfide bonds between individual heparan sulfate proteoglycan monomers may account for the large variation in sizes which has been reported for heparan sulfate proteoglycans isolated from a variety of cells and tissues with a variety of extraction procedures.     

Microbial subversion of heparan sulfate proteoglycans

The interactions between the host and microbial pathogen largely dictate the onset, progression, and outcome of infectious diseases. Pathogens subvert host components to promote their pathogenesis and, among these, cell surface heparan sulfate proteoglycans are exploited by many pathogens for their initial attachment and subsequent cellular entry. The ability to interact with heparan sulfate proteoglycans is widespread among viruses, bacteria, and parasites. Certain pathogens also use heparan sulfate proteoglycans to evade host defense mechanisms. These findings suggest that heparan sulfate proteoglycans are critical in microbial pathogenesis, and that heparan sulfate proteoglycan-pathogen interactions are potential targets for novel prophylactic and therapeutic approaches.     

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells

Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain.     

硫酸软骨素蛋白多糖与神经系统的发育和再生

硫酸软骨素蛋白多糖（chondroitin sulfate proteoglycans,CSPGs）是中枢神经系统（CNS）细胞外基质中的重要组成成分,在CNS的发育、成熟后正常功能的维持中发挥重要功能,如发育中影响神经细胞的迁移和轴突生长,成年后参与神经可塑性的控制等;而病理条件下,如CNS受损后又可做为胶质瘢痕的重要组分抑制受损神经的再生。研究发现,用酶降解CSPGs的糖氨多糖链或阻断其合成可以有效地削弱CSPGs对受损神经的抑制作用,促进轴突再生。然而,精确调控CSPGs特定时空表达模式的分子机制,以及功能发挥所涉及的完整信号转导通路还有待进一步研究。     

Chondroitin sulfate proteoglycans in neural development and regeneration

Proteoglycans are of two main types, chondroitin sulfate (CSPGs) and heparin sulfate (HSPGs). The CSPGs act mainly as barrier-forming molecules, whereas the HSPGs stabilise the interactions of receptors and ligands. During development CSPGs pattern cell migration, axon growth pathways and axon terminations. Later in development and in adulthood CSPGs associate with some classes of neuron and control plasticity. After damage to the nervous system, CSPGs are the major axon growth inhibitory component of the glial scar tissue that blocks successful regeneration. CSPGs have a variety of roles in the nervous system, including binding to molecules and blocking their action, presenting molecules to cells and axons, localising active molecules to particular sites and presenting growth factors to their receptors.     

硫酸肝素蛋白多糖在疾病中的作用

硫酸肝素蛋白多糖广泛分布于动物组织的细胞膜和细胞外基质,对于机体发育和维持生理平衡至关重要.聚糖链硫酸肝素特有的分子结构使得这类大分子复合物具有多种生物功能,这些功能主要通过与蛋白质配体的结合实现.细胞表面的硫酸肝素蛋白多糖介导多种细胞活性因子与其受体的结合,参与信号转导的过程.硫酸肝素蛋白多糖也是细胞间质的重要组成部分,与胶原蛋白一起维持间质结构的稳定.肝素酶通过降解硫酸肝素从而调节细胞因子的活性和细胞间质的微环境.因此,揭示硫酸肝素的分子结构及其功能是生物学的一个重要研究方向.然而,由于硫酸肝素结构复杂,且不均一,使得这个领域的研究发展相对缓慢.不过,随着分析手段的提高和完善,国际上对于硫酸肝素结构与功能的报道迅速增加,同时国内对于硫酸肝素的研究也逐步受到重视.关于硫酸肝素的生理功能最近已有几篇比较全面的综述.此综述主要介绍硫酸肝素在病变中的作用,旨在探讨利用硫酸肝素和肝素酶作为靶标,研发预防和治疗这些疾病药物的可能性.     

Biology of cell surface heparan sulfate proteoglycans

The central question in cell biology is how cells detect, interact and respond to extracellular matrix. The cell surface molecules, which mediate this recognition, consist of a lipophilic membrane domain and an ectodomain binding matrix materials. One group of this kind of molecules is the cell surface heparan sulfate proteoglycans (HSPG). This review summarizes recent information obtained on the cell surface PG of mouse mammary epithelial cells. The glycosaminoglycan containing ectodomain of this PG binds with high affinity Type I, III and V collagen fibrils and the C-terminal heparin binding domain of fibronectin. The PG is mobile on the cell surface, but can be immobilised by ligand binding. At the same time the PG associates with cytoskeleton and links the epithelial cytoskeleton to extracellular matrix. Thus the PG can mediate the changes in the matrix into changes in cellular behaviour, often seen during the regulation of cell shape, proliferation and differentiation. The cell surface PG is also released from the cell surface by cleaving the matrix-binding ectodomain from the membrane domain. Because of the binding properties of the ectodomain, this shedding may provide a means by which epithelial cells loosen their association with the matrix and with other cells, e.g., during normal epithelial development and the invasion of carcinomas.     

Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans

Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production.     

Altered synthesis of heparan sulfate proteoglycans at low sulfate concentration

The structural alterations in heparan sulfate produced by sulfate deprivation were studied in cell cultures of the Engelbreth-Holm-Swarm tumor. Tumor cells were labeled in vitro with [3H]glucosamine and/or [35S]sulfate in media containing either 300 microM MgSO4 or no added carrier sulfate, and the newly synthesized proteoglycans isolated by chromatography on DEAE-Sephacel. The proteoglycans isolated from low sulfate cultures showed a reduced affinity for the column eluting at lower salt concentrations compared with the proteoglycans isolated from cultures containing sulfate, suggesting that the former were undersulfated. Analysis of the isolated heparan sulfate side chains indicated that two pools of heparan sulfate were present which differed in their degree of sulfation. Both pools were synthesized by both high sulfate and low sulfate cultures, but the highly sulfated pool was the predominant form produced in sulfate containing cultures, while the undersulfated pool was the predominant form synthesized in low sulfate cultures. The more sulfated pool contained more N-sulfate than the less sulfated pool. Few if any free amino groups were detected in either pool, suggesting that the initial deacetylation step in the biosynthesis of heparan sulfate is tightly coupled to the N-sulfation step in the cells.     

Occurrence of the HNK-1 epitope (3-sulfoglucuronic acid) in PC12 pheochromocytoma cells, chromaffin granule membranes, and chondroitin sulfate proteoglycans

After biosynthetic labeling of sulfated glycoproteins in rat and goldfish brain and PC12 pheochromocytoma cells with sodium [35S]sulfate, it was observed that all of the bands reactive with the HNK-1 antibody on immunoblots of sodium dodecyl sulfate-polyacrylamide gels corresponded with sulfate-labeled proteins detected by fluorography. These results support data from other studies, which indicate that the HNK-1 epitope is a 3-sulfo-glucuronic acid residue. In addition to its presence in a wide range of nervous tissue glycoproteins, the HNK-1 epitope was also detected in chromaffin granule membranes, chondroitinase ABC, and in chondroitin sulfate proteoglycans of brain, cartilage, and chondrosarcoma. However, it is not present in the heparan sulfate proteoglycan of brain, or in either of two chondroitin sulfate/dermatan sulfate proteoglycans in the chromaffin granule matrix.     

Developmental roles of heparan sulfate proteoglycans in Drosophila

The formation of complex patterns in multi-cellular organisms is regulated by a number of signaling pathways. In particular, the Wnt and Hedgehog (Hh) pathways have been identified as critical organizers of pattern in many tissues. Although extensive biochemical and genetic studies have elucidated the central components of the signal transduction pathways regulated by these secreted molecules, we still do not understand fully how they organize gradients of gene activities through field of cells. Studies in Drosophila have implicated a role for heparan sulfate proteoglycans (HSPGs) in regulating the signaling activities and distribution of both Wnt and Hh. Here we review these findings and discuss various models by which HSPGs regulate the distributions of Wnt and Hh morphogens. Published in 2003.     

CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans

The chemokine CXCL12 (also known as stromal cell-derived factor, SDF-1) is constitutively expressed by stromal resident cells and is involved in the homeostatic and inflammatory traffic of leukocytes. Binding of CXCL12 to glycosaminoglycans on endothelial cells (ECs) is supposed to be relevant to the regulation of leukocyte diapedesis and neoangiogenesis during inflammatory responses. To improve our understanding of the relevance of this process to rheumatoid arthritis (RA), we have studied the mechanisms of presentation of exogenous CXCL12 by cultured RA ECs. RA synovial tissues had higher levels of CXCL12 on the endothelium than osteoarthritis (OA) tissues; in both, CXCL12 colocalized to heparan sulfate proteoglycans (HSPGs) and high endothelial venules. In cultured RA ECs, exogenous CXCL12α was able to bind in a CXCR4-independent manner to surface HSPGs. Desulfation of RA EC HSPGs by pretreatment with sodium chlorate, or by replacing in a synthetic CXCL12α the residues Lys24 and Lys27 by Ser (CXCL12α-K2427S), decreased or abrogated the ability of the chemokine to bind to RA ECs. Ex vivo, synovial ECs from patients with either OA or RA displayed a higher CXCL12-binding capacity than human umbilical vein ECs (HUVECs), and in HUVECs the binding of CXCL12 was increased on exposure to tumor necrosis factor-α or lymphotoxin-α₁β₂. Our findings indicate that CXCL12 binds to HSPGs on ECs of RA synovium. The phenomenon relates to the interaction of HSPGs with a CXCL12 domain with net positive surface charge located in the first β strand, which encompasses a canonical BXBB HSPG-binding motif. Furthermore, we show that the attachment of CXCL12 to HSPGs is upregulated by inflammatory cytokines. Both the upregulation of a constitutive chemokine during chronic inflammation and the HSPG-dependent immobilization of CXCL12 in EC surfaces are potential sites for therapeutic intervention.     

Proteomic analysis of rat pheochromocytoma PC12 cells

PC12 cell line is well documented and widely applied as many kinds of models in neurobiological and neurochemical studies. Yet a thorough proteomic analysis has not been performed so far. Here we report the construction of a large-scale 2-D protein database for PC12 cells. The proteins extracted from PC12 cells were separated by 2-DE and identified by MALDI-TOF/TOF MS. A total of 1080 protein spots, excised from three different 2-D gels, were identified with high confidence. These proteins represent 474 different gene products, mainly binding proteins and enzymes. Three hundred and seven identified protein spots were located in the low-molecular weight region below 20 kDa. This database today represents one of the largest 2-D databases for higher eukaryotic cell proteomes and for low-molecular weight proteins. In addition, fragment ion spectra obtained by TOF/TOF confirmed that calcylin in PC12 cells was N-acetylated. The database of PC12 proteome is expected to be a powerful tool for neuroscientists.