Kegg Pathway: Chondroitin sulfate biosynthesis

J Biol Chem. 2008 Aug 14;
Garud DR, Tran VM, Victor XV, Koketsu M, Kuberan B

Proteoglycans are composed of a protein moiety and a complex glycosaminoglycan (GAG) polysaccharide moiety. GAG chains are responsible for various biological activities. GAG chains are covalently attached to serine residues of the core protein. The first step in proteoglycan biosynthesis is xylosylation of certain serine residues of the core protein. A specific linker tetrasaccharide is then assembled and serves as an acceptor for elongation of GAG chains. If the production of endogenous GAG chains is selectively inhibited, one could determine the role of these endogenous molecules in physiological and developmental functions in a spatiotemporal manner. biosynthesis of proteoglycans are often blocked with the aid of non-specific agents such as chlorate, a bleaching agent, and brefeldin A, a fungal metabolite, to elucidate the biological roles of GAG chains. Unfortunately, these agents are highly lethal to model organisms. Xylosides are known to prime GAG chains. Therefore, we hypothesized that modified xylose analogues may able to inhibit the biosynthesis of proteoglycans. To test this, we synthesized a library of novel 4-deoxy-4-fluoro-xylosides with various aglycones using click chemistry and examined each for their ability to inhibit heparan sulfate and Chondroitin sulfate using CHO cells as a model cellular system.

Biotech Histochem. 2008 Feb; 83(1): 47-53
Melrose J, Smith SM, Smith MM, Little CB

Histochoice is a proprietary nontoxic, non-cross-linking fixative designed by the manufacturer to replace formaldehyde based fixation protocols. We compared Histochoice and formalin fixation for several cartilaginous tissues including, articular and growth plate cartilage, meniscus and intervertebral disc. The tissues were stained with general histology stains including toluidine blue for tissue proteoglycans, picrosirius red to evaluate collagenous organization, and hematoxylin and eosin to assess cell morphology. The Chondroitin sulfate and heparin sulfate substituted proteoglycans aggrecan and perlecan were also immunolocalized in some of the tissues to provide a comparison. Histochoice did not fix deep into the tissue blocks resulting in focal loss of aggrecan and other matrix components from the more central regions of the blocks. This was evident in toluidine blue stained sections of immature tibial articular cartilage where loss of glycosaminoglycan was significant in Histochoice fixed tissues. Histochoice fixation worked well, however, in the aggrecan and perlecan immunohistology applications where its non-cross-linking traits were conducive to epitope retrieval and identification by primary antibodies to extracellular matrix components.

Ann Otol Rhinol Laryngol. 2008 May; 117(5): 371-81
Hahn MS, Jao CY, Faquin W, Grande-Allen KJ

OBJECTIVES: This study was designed to quantify the specific glycosaminoglycans (GAGs) in the midmembranous vocal fold (VF) lamina propria (LP) and to interpret their presence in relation to the known stresses borne by each LP layer. METHODS: GAGs from normal human LP and from both normal and scarred canine LPs were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). Immunostaining was conducted to give insight into the spatial distribution of each GAG type. RESULTS: Hyaluronan composes roughly 0.64% +/- 0.41% of the human LP as measured relative to tissue total protein. Chondroitin sulfate and/or dermatan sulfate (CS/DS), keratan sulfate, and heparan sulfate chains constitute approximately 23.9% +/- 12.1%, 14.7% +/- 6.1%, and 61.4% +/- 13.6%, respectively, of human LP sulfated GAGs. CONCLUSIONS: Observed CS/DS sulfation patterns imply that versican is a major contributor to human LP CS levels. In addition, examination of LP GAG with respect to gender revealed a significant variation in total levels of CS/DS and a potential difference in the levels of versican relative to decorin and biglycan. In dogs, LP scarring appeared to result in a reduction in hyaluronan and CS/DS. These FACE results were combined with histologic data to update current descriptive models linking LP microstructure with the regional variations in LP loading.

Am J Pathol. 2008 Jul; 173(1): 77-92
Beck H, Semisch M, Culmsee C, Plesnila N, Hatzopoulos AK

Ischemic brain injury causes tissue damage and neuronal death. The deficits can often be permanent because adult neurons fail to regenerate. One barrier to neuronal regeneration is the formation of the glial scar, a repair mechanism that is otherwise necessary to seal off necrotic areas. The process of gliosis has been well described, but the mechanisms regulating the robust production of scar components after injury remain poorly understood. Here we show that the early growth response 1 transcriptional factor (Egr-1, also called Krox24, Zif268, and NGFI-A) is expressed in astrocytes in the ventricular wall, corpus callosum, and striatum of normal mouse brain. After experimental stroke caused by permanent occlusion of the middle cerebral artery, Egr-1 was expressed long term in reactive astrocytes that accumulate around the injury site. Gain- and loss-of-function studies in primary astrocytes indicated that Egr-1 regulates the transcription of Chondroitin sulfate proteoglycans genes, the main extracellular matrix proteins of the glial scar. Egr-1 bound to a site within the phosphacan promoter and transactivated its expression. Egr-1-deficient mice accumulated lower levels of phosphacan RNA and protein than wild-type mice after stroke, but there were no measurable differences in neurite outgrowth toward the infarct area between the two groups. Our findings suggest that Egr-1 is an important component of the transcriptional network regulating genes involved in gliosis after ischemic injury.

Development. 2008 Jul; 135(13): 2215-20
Sohaskey ML, Yu J, Diaz MA, Plaas AH, Harland RM

Properly positioned synovial joints are crucial to coordinated skeletal movement. Despite their importance for skeletal development and function, the molecular mechanisms that underlie joint positioning are not well understood. We show that mice carrying an insertional mutation in a previously uncharacterized gene, which we have named Jaws (joints abnormal with splitting), die perinatally with striking skeletal defects, including ectopic interphalangeal joints. These ectopic joints develop along the longitudinal axis and persist at birth, suggesting that JAWS is uniquely required for the orientation and consequent positioning of interphalangeal joints within the endochondral skeleton. Jaws mutant mice also exhibit severe chondrodysplasia characterized by delayed and disorganized maturation of growth plate chondrocytes, together with impaired Chondroitin sulfation and abnormal metabolism of the Chondroitin sulfate proteoglycan aggrecan. Our findings identify JAWS as a key regulator of chondrogenesis and synovial joint positioning required for the restriction of joint formation to discrete stereotyped locations in the embryonic skeleton.

Malar J. 2008; 7: 104
Resende M, Nielsen MA, Dahlbäck M, Ditlev SB, Andersen P, Sander AF, Ndam NT, Theander TG, Salanti A

BACKGROUND: Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes binding the placental receptor Chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule. METHODS: To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes. RESULTS: The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes. CONCLUSION: Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.

N Engl J Med. 2008 Jun 5; 358(23): 2505-9
Schwartz LB

Mol Vis. 2008; 14: 975-82
Zheng S, Yin ZQ, Zeng YX

PURPOSE: To investigate the distribution, expression, and activity of tissue plasminogen activator (tPA) in the visual cortex of the Long Evans rat during postnatal development, and to explore the relationship between tPA levels and the critical period of visual cortical plasticity. METHODS: Long Evans rats of either sex (n=131) were divided by postnatal age in weeks (PW) into five groups: PW1 (6-8 days, before eye opening, n=19), PW3 (20-22 days, beginning of critical period, n=28), PW5 (34-36 days, later stage of critical period, n=28), PW7 (48-50 days, end of critical period, n=28), and PW14 (95-100 days, adult, n=28). The distribution and expression of tPA was detected using immunofluorescence histochemistry and western blot analysis, respectively. tPA activity in the visual cortex was determined using a chromogenic assay kit. RESULTS: tPA-containing cells were mostly located in visual cortex layer II-III and layer IV during postnatal development. In layer II-III the density of tPA-containing cells reached peak at PW 5, and then reduced to minimum at PW14. In layer IV and V-VI, the density of tPA-containing cells reached a maximum at PW3, and then decreased to the minimum at PW14. Western blot analysis indicated that tPA was detected in visual cortex of rats from PW3 onwards with the highest quantity present at PW5. By comparison, the peak in tPA activity occurred slightly earlier at PW3, and then decreased steadily to lower levels at PW14. CONCLUSIONS: The critical period of visual cortical plasticity, which occurs in early postnatal life, correlates well with tPA expression in the rat visual cortex. This suggests that the expression of tPA is produced in sufficient amounts to balance the increase of Chondroitin sulfate proteoglycan expression, at the same time blocking its function, thus allowing synaptic modification to continue. tPA activity may be one of the factors influencing the duration of the critical period and underlying the heterogeneity of synaptic plasticity between visual cortex layer II-III and layer IV.

Invest Ophthalmol Vis Sci. 2008 Jun; 49(6): 2495-505
Keller KE, Bradley JM, Kelley MJ, Acott TS

PURPOSE: Glycosaminoglycans (GAGs) have been implicated in the regulation of outflow resistance of aqueous humor flow through the trabecular meshwork (TM). Their role was further investigated by assessment of the effects of chlorate, an inhibitor of sulfation, and beta-xyloside, which provides a competitive nucleation point for addition of disaccharide units, in anterior segment perfusion culture. METHODS: Outflow facility was measured in perfused porcine and human anterior organ cultures treated with 20 or 50 mM sodium chlorate, or 1 mM beta-xyloside. Perturbation of extracellular matrix (ECM) components was assessed in paraffin-embedded sections by immunofluorescence and confocal microscopy. Parallel experiments were conducted on cultured TM cells. RESULTS: Outflow facility increased in porcine eyes with chlorate (3-fold) and beta-xyloside (3.5-fold) treatments. In human eyes, outflow increased approximately 1.5-fold and took longer (>48 hours) to occur. By confocal microscopy, immunostaining for Chondroitin and heparan sulfates was observed on edges of human TM beams in nontreated eyes, with intense staining in the juxtacanalicular tissue (JCT) region. In treated eyes, staining of beam edges was severely reduced and was instead found in plaques. Chlorate treatment resulted in a striated pattern of GAG staining in the human JCT region. Fibronectin immunostaining was altered in beta-xyloside-treated eyes, whereas in cell culture, chlorate induced formation of thick fibronectin fibrils, to which tenascin C colocalized. CONCLUSIONS: Disrupting GAG chain biosynthesis increased outflow facility in perfusion culture and induced atypical ECM molecule interactions in cell culture. This study provides direct evidence of the critical role of GAG chains in regulating outflow resistance in human TM.

Am J Hum Genet. 2008 Jun; 82(6): 1368-74
Hermanns P, Unger S, Rossi A, Perez-Aytes A, Cortina H, Bonafé L, Boccone L, Setzu V, Dutoit M, Sangiorgi L, Pecora F, Reicherter K, Nishimura G, Spranger J, Zabel B, Superti-Furga A

Deficiency of carbohydrate sulfotransferase 3 (CHST3; also known as Chondroitin-6-sulfotransferase) has been reported in a single kindred so far and in association with a phenotype of severe chondrodysplasia with progressive spinal involvement. We report eight CHST3 mutations in six unrelated individuals who presented at birth with congenital joint dislocations. These patients had been given a diagnosis of either Larsen syndrome (three individuals) or humero-spinal dysostosis (three individuals), and their clinical features included congenital dislocation of the knees, elbow joint dysplasia with subluxation and limited extension, hip dysplasia or dislocation, clubfoot, short stature, and kyphoscoliosis developing in late childhood. Analysis of Chondroitin sulfate proteoglycans in dermal fibroblasts showed markedly decreased 6-O-sulfation but enhanced 4-O-sulfation, confirming functional impairment of CHST3 and distinguishing them from diastrophic dysplasia sulphate transporter (DTDST)-deficient cells. These observations provide a molecular basis for recessive Larsen syndrome and indicate that recessive Larsen syndrome, humero-spinal dysostosis, and spondyloepiphyseal dysplasia Omani type form a phenotypic spectrum.

Anal Chim Acta. 2008 Jun 23; 618(2): 218-26
Bouças RI, Trindade ES, Tersariol IL, Dietrich CP, Nader HB

sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, Chondroitin 4-sulfate, Chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by Chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.

Int J Cosmet Sci. 1998 Jun; 20(3): 159-73
Frei V, Perrier E, Orly I, Huc A, Augustin C, Damour O

Skin firmness, elasticity and tone are gradually lost with age. These changes originate in the dermis and correspond to a decrease in the ability of cells, particularly the fibroblasts, to regenerate the molecules which make up the extracellular matrix. Skin ageing is also characterized by a reduction of the epidermal thickness and by a flattening of the basal membrane. The recent development of two 3-dimensional culture systems, in which the cells develop within a porous structure reproducing the extracellular matrix of the human dermis, is a way of reproducing in vivo conditions and demonstrating the biological effects of anti-ageing compounds. The dermal equivalent model used in this study is composed of a dermal matrix made of collagen-chitosan-glycosaminoglycans populated by normal human fibroblasts which synthesized their own extracellular matrix. A skin equivalent model is obtained by the cell culture of normal human keratinocytes onto a dermal equivalent elevated at the air-liquid interface. Such models were used to prove anti-ageing activity of promising compounds. Cosmetic Science has used many protein hydrolysates in order to fight skin ageing, but up to now, these natural peptides were poorly studied, and their efficacy poorly demonstrated. Eight protein hydrolysates were screened in a proliferation study in monolayered cultures giving two selected polypeptides. A soya derived peptide was used for an efficiency study in 3-dimensional models. In the dermal equivalent model, this peptide increased fibroblast proliferation by 40% and led to a stimulation of collagen formation (165%) and elastin (116%) synthesis. The effect of this soya peptide on glycosaminoglycan synthesis was also significant, with increases of 36% for Chondroitin-4-sulfate and 68% for hyaluronic acid. These results were confirmed using a skin equivalent model. In this model, the soya peptide increased the thickness of the epidermis.

Coll Antropol. 2008 Mar; 32(1): 201-7
Katusić A, Jurić-Lekić G, Jovanov-Milosević N, Vlahović M, Jezek D, Serman L, Sincić N, Veljanovska B, Bulić-Jakus F

Investigation of the developmental potential of immature tissues is important for novel approaches to human regenerative medicine. Development of the fetal neural retina has therefore been investigated in two experimental systems. Retinas were microsurgically isolated from 20-days-old rat fetuses and cultivated in vitro for 12 days or transplanted in vivo under the kidney capsule of adult males for as long as 6 months. Shedding of the photoreceptor outer segment which is a process occurring at the terminal stage of photoreceptor differentiation was observed in culture by transmission electron microscopy (TEM). In transplants, no photoreceptors were found although markers of terminal neural and glial differentiation (e,g. synaptophysin, chromogranin and glial fibrilary acidic protein--GFAP) along with the molecules involved in the process of differentiation (guidance molecule semaphorin IIIA and Chondroitin sulfate proteoglycan) were expressed. Semaphorin was differentially expressed being absent from older transplants. Proliferating cell nuclear antigen and nestin (marker of undifferentiated neural cells) were still weakly expressed even in six-months-old transplants. We could conclude that in both our experimental systems fetal neural retina proceeded to differentiate further on. However, even in long-term ectopic transplants a small population of cells still retained the potential for proliferation and has not yet reached the stage of terminal differentiation.

Infect Immun. 2008 Jun; 76(6): 2808; author reply 2808-9
Beeson JG

Int Immunopharmacol. 2008 Jul; 8(7): 1056-8
Loeb LM, Naffah-Mazzacoratti MG, Porcionatto MA, Martins JR, Kouyoumdjian M, Weckx LM, Nader HB

Glossodynia or burning mouth syndrome is a multifunctional disorder. The oral mucosa is apparently normal but patients report burning and dried mouth and painful tongue and lips. The present study reports biochemical and physiological markers in saliva of patients presenting glossodynia compared to normal subjects. Saliva-buffering capacity and contents of protein and hyaluronic (HA) acid were similar in both groups. In contrast, Chondroitin sulfate (CS) concentration was decreased in the saliva of patients with glossodynia when compared to control group (p=0.0036). On the other hand glandular kallikrein showed increased activity in the saliva of patients compared to normal subjects (p<0.0001). The data suggest involvement of the kinin system, possibly related to the low levels of CS. Depression could explain the low level of serotonin in patient serum (p=0.0478).

Surg Neurol. 2008 Jun; 69(6): 568-77; discussion 577
Shields LB, Zhang YP, Burke DA, Gray R, Shields CB

BACKGROUND: Chondroitin sulfate proteoglycans are up-regulated in the spinal cord after SCI, creating a molecular barrier inhibitory to axon growth. Chondroitinase ABC degrades CSPGs in vitro and in vivo. METHODS: We studied whether IT ChABC promotes axonal regeneration in a laceration model of SCI. Three groups of Sprague-Dawley rats were used: control and rats treated with low-dose and high-dose IT ChABC. Chondroitin sulfate proteoglycan breakdown products were measured by 2-B-6 expression, and intact CSPGs by CS-56 expression. Sensory axonal regeneration was traced after CTB injection into the median, ulnar, and sciatic nerves. RESULTS: CS-56 expression was down-regulated and 2-B-6 expression was increased in the groups treated with IT ChABC but not in the control. Laminin and GFAP immunoreactivity was unaltered in the ChABC groups. The number of axons growing into the scar was 3.1 times greater (P < .01) in the high-dose ChABC group and 2.1 times greater (P < .01) in the low-dose group compared with the controls. The length of axonal growth after high- and low-dose ChABC was 9.9 (P < .01) and 8.3 (P < .01) times greater, respectively, than in the control group. Axons extended across the lesion gap and into the distal spinal cord stump in 2 of 8 (low dose) and in 3 of 9 (high dose) rats compared with none in the control group. CONCLUSIONS: Intrathecal ChABC administration caused a slight decrease in CSPGs in the scar after a laceration SCI with a minimal increase in sensory axonal regeneration into and across the laceration gap.

Respir Res. 2008; 9: 41
Merrilees MJ, Ching PS, Beaumont B, Hinek A, Wight TN, Black PN

BACKGROUND: COPD is characterised by loss of alveolar elastic fibers and by lack of effective repair. Elastic fibers are assembled at cell surfaces by elastin binding protein (EBP), a molecular chaperone whose function can be reversibility inhibited by Chondroitin sulphate of matrix proteoglycans such as versican. This study aimed to determine if alveoli of patients with mild to moderate COPD contained increased amounts of versican and a corresponding decrease in EBP, and if these changes were correlated with decreases in elastin and FEV1. METHODS: Lung samples were obtained from 26 control (FEV1 > or = 80% predicted, FEV1/VC >0.7) and 17 COPD patients (FEV1 > or = 40% - <80% predicted, FEV1/VC < or = 0.7) who had undergone a lobectomy for bronchial carcinoma. Samples were processed for histological and immuno-staining. Volume fractions (Vv) of elastin in alveolar walls and alveolar rims were determined by point counting, and versican and EBP assessed by grading of staining intensities. RESULTS: Elastin Vv was positively correlated with FEV1 for both the alveolar walls (r = 0.66, p < 0.001) and rims (r = 0.41, p < 0.01). Versican was negatively correlated with FEV1 in both regions (r = 0.30 and 0.32 respectively, p < 0.05), with the highest staining intensities found in patients with the lowest values for FEV1. Conversely, staining intensities for EBP in alveolar walls and rims and were positively correlated with FEV1 (r = 0.43 and 0.46, p < 0.01). CONCLUSION: Patients with mild to moderate COPD show progressively increased immuno-staining for versican and correspondingly decreased immuno-staining for EBP, with decreasing values of FEV1. These findings may explain the lack of repair of elastic fibers in the lungs of patients with moderate COPD. Removal of versican may offer a strategy for effective repair.

Glycobiology. 2008 Aug; 18(8): 602-14
Ishii M, Maeda N

Chondroitin sulfate (CS) proteoglycans are major components of the cell surface and the extracellular matrix in the developing brain and bind to various proteins via CS chains in a CS structure-dependent manner. This study demonstrated the expression pattern of three CS sulfotransferase genes, dermatan 4-O-sulfotransferase (D4ST), uronyl 2-O-sulfotransferase (UST), and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), in the mouse postnatal cerebellum. These sulfotransferases are responsible for the biosynthesis of oversulfated structures in CS chains such as B, D, and E units, which constitute the binding sites for various heparin-binding proteins. Real-time reverse transcription-polymerase chain reaction analysis indicated that the expression of UST increased remarkably during cerebellar development. The amounts of B and D units, which are generated by UST activity, in the cerebellar CS chains also increased during development. In contrast, the expression of GalNAc4S-6ST and its biosynthetic product, E unit, decreased during postnatal development. In situ hybridization experiments revealed the levels of UST and GalNAc4S-6ST mRNAs to correlate inversely in many cells including Purkinje cells, granule cells in the external granular layer, and inhibitory interneurons. In these neurons, the expression of UST increased and that of GalNAc4S-6ST decreased during development and/or maturation. D4ST was also expressed by many neurons, but its expression was not simply correlated with development, which might contribute to the diversification of CS structures expressed by distinct neurons. These results suggest that the CS structures of various cerebellar neurons change during development and such changes of CS are involved in the regulation of various signaling pathways.

Cell Transplant. 2008; 17(1-2): 91-7
Ueda M, Matsumoto S, Hayashi S, Kobayashi N, Noguchi H

Although islet transplantation is a promising therapeutic option for the treatment of type 1 diabetes, the shortage of suitable donor tissues remains a major obstacle. Pancreatic stem/progenitor cells residing within the ductal epithelium have been used to generate human islet-like clusters, but there is no efficient strategy for facilitating differentiation of progenitor cells into insulin-producing cells. A previous study reported that exogenous PDX-1 protein can be transduced into pancreatic stem/progenitor cells and induce differentiation of the cells into insulin-producing cells without requiring gene transfer technology. This study provides genetic and biochemical evidence that cell membrane heparan sulfate proteoglycans are required for extracellular PDX-1 internalization. Heparin, one of the soluble glycosaminoglycans (GAGs), inhibited PDX-1 internalization, while Chondroitin sulfate A, B, and C caused only very limited inhibition. Cell treatment with heparinase-III demonstrated impaired PDX-1 internalization, while treatment with Chondroitinase ABC, or with Chondroitinase AC, was completely ineffective in inhibiting PDX-1 internalization. Different mutant cell lines originating from CHO K1 cells and defective in GAG biosynthesis were also examined. PDX-1 internalization was significantly reduced in both pgs A-745 mutant cells, which are defective in a enzyme that initiates GAG synthesis, and pgs B-618 cells, which produce about 15% of the amount of GAGs synthesized by wild-type cells. These data indicate that cell-surface heparan sulfate proteoglycans are required for PDX-1 internalization and that PDX-1 protein transduction could be a valuable strategy for inducing insulin expression in pancreatic stem/progenitor cells without requiring gene transfer technology.

J Infect Dis. 2008 Apr 15; 197(8): 1119-23
Oleinikov AV, Francis SE, Dorfman JR, Rossnagle E, Balcaitis S, Getz T, Avril M, Gose S, Smith JD, Fried M, Duffy PE

The variant surface antigen VAR2CSA is a pregnancy malaria vaccine candidate, but its size and polymorphism are obstacles to development. We expressed 3D7-type VAR2CSA domains in Escherichia coli as insoluble His-tagged proteins (Duffy binding-like [DBL] domains DBL1, DBL3, DBL4, and DBL5) that were denatured and refolded or as soluble glutathione S-transferase-tagged protein (DBL6). Anti-DBL5 antiserum cross-reacted with surface proteins of Chondroitin sulfate A (CSA)-binding laboratory strains (3D7-CSA and FCR3-CSA) and a clinical pregnancy malaria isolate, whereas anti-DBL6 antiserum reacted only to 3D7 surface protein. This is the first report that E. coli-expressed VAR2CSA domains induce antibody to native VAR2CSA.