Hans Kresse

[46] Enzymic diagnosis of the genetic mucopolysaccharide storage disorders

Publisher Summary This chapter presents procedure for enzymic diagnosis of genetic mucopolysaccharide storage disorders particularly two enzyme defects underlying the Morquio syndrome. For the diagnosis of the rare disorders, fibroblasts are the most convenient enzyme source because of the possibility of performing extensive investigations and because of the relatively high activity found in normal cells. However, enzymic diagnosis can be performed also on leukocytes. α-glucosaminide N-acetyltransferase can be measured in cultured fibroblasts or amniotic fluid cells, leukocytes, and tissues, which is based on the principle that states the substrate, a trisaccharide with the structure O- (α-D-2-amino-2-deoxyglucopyranosyl)-(1 →4)-O-(β-D-glucopyranosyluronicacid)-(1→4)-2,5-anhydro-D-[ 3 H]mannitol, prepared from heparin, can be N-acetylated by acetyl-CoA:α-glucosaminide N-acetyltransferase in the presence of acetyl-CoA. The product bearing an N-acetylated glucosamine residue at the nonreducing terminal, can be hydrolyzed by α-N acetylglucosaminidase to N-acetylglucosamine and a radioactive disaccharide, which can be further split by β-glucuronidase. The positively charged substrate is separated from the neutral or negatively charged products by passage over a cation-exchange resin.

Absence of Decorin Adversely Influences Tubulointerstitial Fibrosis of the Obstructed Kidney by Enhanced Apoptosis and Increased Inflammatory Reaction

Decorin, a small dermatan-sulfate proteoglycan, participates in extracellular matrix assembly and influences directly and indirectly cell behavior via interactions with signaling membrane receptors and transforming growth factor (TGF)-beta. We have therefore compared the development of tubulointerstitial kidney fibrosis in wild-type (WT) and decorin-/- mice in the model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin-/- mice did not differ in any aspect from their WT counterparts. However, already 12 hours after obstruction decorin-/- animals showed lower levels of p27(KIP1) and soon thereafter a more pronounced up-regulation and activation of initiator and effector caspases followed by enhanced apoptosis of tubular epithelial cells. Later, a higher increase of TGF-beta1 became apparent. After 7 days, there was an up to 15-fold transient up-regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan-expressing mononuclear cells. Other small proteoglycans showed no similar response. Because of enhanced degradation of type I collagen, end-stage kidneys from decorin-/- animals were more atrophic than WT kidneys. These data suggest that decorin exerts beneficial effects on tubulointerstitial fibrosis, primarily by influencing the expression of a key cyclin-dependent kinase inhibitor and by limiting the degree of apoptosis, mononuclear cell infiltration, tubular atrophy, and expression of TGF-beta1.

Selective inactivity of TGF-β/decorin complexes

Previous studies had shown that binding of TGF‐β to the small proteoglycan decorin results in its inactivation. Indeed, in osteosarcoma cells the addition of decorin prevented the TGF‐β1‐mediated up‐regulation of biglycan synthesis. However, the down‐regulation of proteoglycan‐100 remained unaltered. Even in the presence of a 100,000‐fold molar excess of decorin, TGF‐β1 was fully active in U937 monocytes with respect to the inhibition of cell proliferation. There was no inhibition of the TGF‐β‐mediated stimulation of the retraction of fibroblast‐populated collagen lattices. Thus, the formation of TGF‐β/decorin complexes leads to the neutralization of distinct effects only.

Small proteoglycans in human diabetic nephropathy: discrepancy between glomerular expression and protein accumulation of decorin, biglycan, lumican, and fibromodulin

Small leucine‐rich proteoglycans (SLRPs), for example, decorin, biglycan, fibromodulin, and lumican, are extracellular matrix organizers and binding partners of TGF‐β. Decorin is also involved in growth control and angiogenesis. Hence, these proteoglycans are likely of importance in the pathogenesis of diabetic glomerulosclerosis. In normal kidney, SLRPs were preferentially expressed in the tubulointerstitium. Weak expression occurred in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, by distal tubular cells and in collecting ducts. In all stages of diabetic nephropathy, there was a marked up‐regulation of the proteoglycans in tubulointerstitium and glomeruli. Decorin and lumican became expressed in tubuli. However, in glomeruli, overexpression was not mirrored by local proteoglycan accumulation except in advanced nephropathy. In severe glomerulosclerosis, increased decorin concentrations were found in plasma and urine, and urinary TGF‐β/decorin complexes could be demonstrated indirectly. The failure to detect an increased glomerular proteoglycan quantity during the development of nephropathy could be explained by assuming that they are secreted into the mesangial matrix, but cleared via the vasculature or the urinary tract, in part as complexes with TGF‐β. They could thereby counteract the vicious circle being characterized by increased TGF‐β production and increased matrix deposition in diabetic nephropathy.

Critical Role of Glutamate in a Central Leucine-rich Repeat of Decorin for Interaction with Type I Collagen

The chondroitin/dermatan sulfate proteoglycan decorin is known to interact via its core protein with fibrillar collagens, thereby influencing the kinetics of fibril formation and the final diameter of the fibrils. To define the binding site(s) for type I collagen along the core protein, which is mainly composed of leucine-rich repeat structures, decorin cDNAs were constructed and expressed in human kidney 293 cells. The constructs encoded (i) C-terminally truncated molecules, (ii) core proteins with deletions of selected leucine-rich repeats, or (iii) various point mutations. The deletion of the sixth leucine-rich repeat Met176–Lys201 and the mutation E180K drastically interfered with the binding to reconstituted type I collagen fibrils. In contrast, the deletion of the seventh repeat Leu202–Ser222 led at the most to a marginally impaired binding, although the secretion of this proteoglycan was abnormally low. Decorin with two other point mutations in the sixth leucine-rich repeat, Lys187 → Gln and Lys200→ Gln, respectively, bound type I collagen either normally or even better than the normal recombinant proteoglycan. These data suggest that a major collagen-binding site of decorin is located within the sixth leucine-rich repeat and that glutamate-180 within this repeat is of special importance for ionic interactions between the two matrix components.

In vivo selective and distant killing of cancer cells using adenovirus-mediated decorin gene transfer

Decorin is a well‐known, ubiquitous proteoglycan that is a normal component of the ECM. Upon transgenic expression of decorin, tumor cells with diverse histogenetic background overexpress p21WAF1, a potent inhibitor of cyclin‐dependent kinase activity, become arrested in G1, and fail to generate tumors in immunocompromised animals. Because decorin is a secreted protein, it has been recently suggested that decorin could act as an autocrine and paracrine regulator of tumor growth. Here, we demonstrate that adenovirus (Ad)‐mediated transfer and expression of human decorin cDNA induced in vivo apoptosis of xenograft tumor cells in nude mice. This oncolytic activity was observed when the Ad vector encoding the decorin cDNA was injected intratumorally (i.t.) or i.v. Importantly, i.t. injection of the decorin Ad vector led to growth inhibition of the injected tumor associated with similar growth inhibition of a distant contralateral tumor, demonstrating a distant decorin antitumoral effect. Immunochemistry against human decorin and decorin quantitation in tumors confirmed that decorin migrated to the tumor distant site. Furthermore, decorin effect was specific to tumor cells, because neither apoptosis nor growth inhibition were observed in nontumoral human cells such as hepatocytes, endothelial cells, and fibroblasts, despite p21 overexpression.

Differential expression of the small chondroitin/dermatan sulfate proteoglycans decorin and biglycan after injury of the adult rat brain.

Chondroitin sulfate proteoglycans are widespread extracellular matrix proteins and are specifically upregulated after CNS injury at the lesion site. Many proteoglycan core proteins have been described in the rat brain, but detailed analysis of individual proteoglycans expressed after injury are missing. The present study represents an initial attempt to assess the diversity and timing of lesion-induced expression of proteoglycans in order to elucidate their functional role in CNS injury and repair. Using immunocytochemical methods we analysed the expression of decorin and biglycan in the transected postcommissural fornix of the adult rat. Transection of the fornix induced the upregulation of both decorin and biglycan. However, their expression differed with respect to time course, regional extent and cellular localization. The rapid upregulation of decorin within a wide area around the lesion was followed by a massive appearance of biglycan that remained restricted to the transection site. Three months after lesion, differences of the area size of decorin- and biglycan-immunoreactivities were no longer detectable. Both proteoglycans were restricted to the lesion site and the fornix stumps. While decorin was primarily expressed by astrocytes, biglycan was deposited extracellularly in sheet-like structures. The upregulation of both proteoglycans persisted for at least up to 6 months after lesion. These strong but divergent lesion-induced expression patterns indicate important but different roles of decorin and biglycan in CNS injury.

Decorin deficiency leads to impaired angiogenesis in injured mouse cornea

Small leucine-rich proteoglycans play important roles in the organization of the extracellular matrix as well as for the regulation of cell behavior; two biological processes that are essential for angiogenesis. We investigated consequences of the targeted ablation of decorin (DCN), biglycan (BGN) and fibromodulin (FMOD) genes on inflammation-induced angiogenesis in the cornea. In wild-type mice, DCN was localized exclusively to the corneal stroma, while FMOD and BGN were more prominently expressed in epithelial cells. Endothelial cells from limbus blood vessels expressed BGN and FMOD, but no DCN. However, after induction of angiogenesis by chemical cauterization, DCN was expressed in the newly formed capillaries, together with BGN and FMOD. Notably, in DCN-deficient mice, the growth of vessels was significantly diminished, whereas it did not significantly change in FMOD- or BGN-deficient animals. Moreover, blood vessels of DCN-deficient mice exhibited a similar expression level of BGN as control mice, while FMOD was increased on day 3 after injury. These results indicate that DCN, in addition to its effects on fibrillogenesis, plays a regulatory role in angiogenesis and that FMOD in endothelial cells may be able to partially substitute for DCN.

Mucopolysaccharidosis III A (Sanfilippo A disease): Deficiency of a heparin sulfamidase in skin fibroblasts and leucocytes

Abstract Cultured skin fibroblasts and peripheral leucocytes from patients with Sanfilippo A disease are strikingly deficient in sulfamidase activity (sulfamatase, EC 3.1.6.?), as measured with heparin - N 35 SO 4 . A partial sulfamidase deficiency was found in the cells of the heterozygote carriers. Since Sanfilippo A fibroblasts have normal sulfate ester hydrolase activities towards oligosaccharides prepared from 35 SO 4 -labelled heparan sulfate by nitrous acid treatment, the basic defect in Sanfilippo A disease is considered to be the inactivity of a heparin (heparan sulfate) sulfamidase.

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.