A solid-phase substrate of heparanase: Its application to assay of human melanoma for heparan sulfate degradative activity

BackgroundDyslipidemia is an important risk factor in CKD. The liver clears triglyceride-rich lipoproteins (TRL) via LDL receptor (LDLR), LDLR-related protein-1 (LRP-1), and heparan sulfate proteoglycans (HSPGs), mostly syndecan-1. HSPGs also facilitate LDLR degradation by proprotein convertase subtilisin/kexin type 9 (PCSK9). Progressive renal failure affects the structure and activity of hepatic lipoprotein receptors, PCSK9, and plasma cholesterol.MethodsUninephrectomy- and aging-induced CKD in normotensive Wistar rats and hypertensive Munich-Wistar-Frömter (MWF) rats.ResultsCompared with 22-week-old sex- and strain-matched rats, 48-week-old uninephrectomized Wistar-CKD and MWF-CKD rats showed proteinuria, increased plasma creatinine, and hypercholesterolemia (all P<0.05), which were most apparent in hypertensive MWF-CKD rats. Hepatic PCSK9 expression increased in both CKD groups (P<0.05), with unusual sinusoidal localization, which was not seen in 22-week-old rats. Heparan sulfate (HS) disaccharide analysis, staining with anti-HS mAbs, and mRNA expression of HS polymerase exostosin-1 (Ext-1), revealed elongated HS chains in both CKD groups. Solid-phase competition assays showed that the PCSK9 interaction with heparin-albumin (HS-proteoglycan analogue) was critically dependent on polysaccharide chain length. VLDL binding to HS from CKD livers was reduced (P<0.05). Proteinuria and plasma creatinine strongly associated with plasma cholesterol, PCSK9, and HS changes.ConclusionsProgressive CKD induces hepatic HS elongation, leading to increased interaction with PCSK9. This might reduce hepatic lipoprotein uptake and thereby induce dyslipidemia in CKD. Therefore, PCSK9/HS may be a novel target to control dyslipidemia.

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Human melanoma associated antigens: A solid-phase assay for detection of specific antibody

Journal of Immunological Methods ◽

10.1016/0022-1759(80)90058-7 ◽

1980 ◽

Vol 39 (3) ◽

pp. 233-246 ◽

Cited By ~ 17

Author(s):

Alton C. Morgan ◽

Darrell R. Galloway ◽

Barry S. Wilson ◽

Ralph A. Reisfeld

Keyword(s):

Specific Antibody ◽

Solid Phase ◽

Human Melanoma ◽

Solid Phase Assay

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A new linker for solid-phase synthesis of heparan sulfate precursors by sequential assembly of monosaccharide building blocks

Chemical Communications ◽

10.1039/c0cc04686h ◽

2011 ◽

Vol 47 (8) ◽

pp. 2390-2392 ◽

Cited By ~ 33

Author(s):

Pawel Czechura ◽

Nerea Guedes ◽

Sebastian Kopitzki ◽

Naiara Vazquez ◽

Manuel Martin-Lomas ◽

...

Keyword(s):

Heparan Sulfate ◽

Solid Phase Synthesis ◽

Solid Phase ◽

Building Blocks ◽

Phase Synthesis ◽

Sequential Assembly

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Adeno-Associated Virus Type 2 Contains an Integrin α5β1 Binding Domain Essential for Viral Cell Entry

Journal of Virology ◽

10.1128/jvi.00843-06 ◽

2006 ◽

Vol 80 (18) ◽

pp. 8961-8969 ◽

Cited By ~ 111

Author(s):

Aravind Asokan ◽

Julie B. Hamra ◽

Lakshmanan Govindasamy ◽

Mavis Agbandje-McKenna ◽

Richard J. Samulski

Keyword(s):

Heparan Sulfate ◽

Virus Type ◽

Solid Phase ◽

Transduction Efficiency ◽

Wild Type ◽

Integrin Α5β1 ◽

Adeno Associated Virus ◽

293 Cells ◽

Α5β1 Integrin

ABSTRACT Integrins have been implicated as coreceptors in the infectious pathways of several nonenveloped viruses. For example, adenoviruses are known to interact with αV integrins by virtue of a high-affinity arginine-glycine-aspartate (RGD) domain present in the penton bases of the capsids. In the case of adeno-associated virus type 2 (AAV2), which lacks this RGD motif, integrin αVβ5 has been identified as a coreceptor for cellular entry. However, the molecular determinants of AAV2 capsid-integrin interactions and the potential exploitation of alternative integrins as coreceptors by AAV2 have not been established thus far. In this report, we demonstrate that integrin α5β1 serves as an alternative coreceptor for AAV2 infection in human embryonic kidney 293 cells. Such interactions appear to be mediated by a highly conserved domain that contains an asparagine-glycine-arginine (NGR) motif known to bind α5β1 integrin with moderate affinity. The mutation of this domain reduces transduction efficiency by an order of magnitude relative to that of wild-type AAV2 vectors in vitro and in vivo. Further characterization of mutant and wild-type AAV2 capsids through transduction assays in cell lines lacking specific integrins, cell adhesion studies, and cell surface/solid-phase binding assays confirmed the role of the NGR domain in promoting AAV2-integrin interactions. Molecular modeling studies suggest that NGR residues form a surface loop close to the threefold axis of symmetry adjacent to residues previously implicated in binding heparan sulfate, the primary receptor for AAV2. The aforementioned results suggest that the internalization of AAV2 in 293 cells might follow a “click-to-fit” mechanism that involves the cooperative binding of heparan sulfate and α5β1 integrin by the AAV2 capsids.

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Proteoglycan involvement during development of lesional pulmonary edema

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.2.l203 ◽

1998 ◽

Vol 274 (2) ◽

pp. L203-L211 ◽

Cited By ~ 20

Author(s):

Daniela Negrini ◽

Alberto Passi ◽

Giancarlo De Luca ◽

Giuseppe Miserocchi

Keyword(s):

Heparan Sulfate ◽

Chondroitin Sulfate ◽

Solid Phase ◽

Guanidine Hydrochloride ◽

Membrane Integrity ◽

Weight Ratio ◽

Chondroitin Sulfate Proteoglycans ◽

Initial Increase ◽

Parietal Pleura ◽

Tissue Compliance

We evaluated the effect of pancreatic elastase (7 IU iv) on pulmonary interstitial pressure (Pip) in in situ rabbit lungs by a micropuncture technique through the intact parietal pleura. Pip was −10.8 ± 2.2 (SD) cmH2O in the control condition, increased to +5.1 ± 1.7 cmH2O at ∼60 min [condition referred to as mild edema (ME)], and subsequently decreased to −0.15 ± 0.8 cmH2O, remaining steady from 80 up to 200 min with a marked increase in lung wet-to-dry weight ratio [condition referred to as severe edema (SE)], suggesting an increase in tissue compliance. We functionally correlated the measured Pip to structural modifications of proteoglycans, the major interfibrillar component of the extracellular matrix (ECM). The strength of the noncovalent bonds linking proteoglycans to other ECM components decreased with increasing severity of edema, as indicated by the increased extractability of proteoglycans with guanidine hydrochloride. Total proteoglycan recovery (expressed as μg hexuronate/g dry tissue) increased from 436.8 ± 14 in the control condition to 495.3 ± 23 and 547.0 ± 10 in ME and SE, respectively. Gel-filtration chromatography showed in ME a fragmentation of heparan sulfate proteoglycans, suggesting that elastase treatment first affected basement membrane integrity, whereas large chondroitin sulfate proteoglycans were degraded only in SE. Elastase caused a fragmentation only of the core protein of proteoglycans, the binding properties of which to collagens, fibronectin, and hyaluronic acid were markedly decreased, as indicated by a solid-phase binding assay. The sequential degradation of heparan sulfate and chondroitin sulfate proteoglycans may account for the initial increase in microvascular permeability, followed by a loss of the native architecture of the ECM, which may be responsible for the increase in tissue compliance.

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