Involvement of heparan sulfate proteoglycans in cellular uptake of high molecular weight kininogen

Abstract

In this study, we analyzed the influence of proteoglycans on the interaction between human high molecular weight kininogen (HK) and the cell surface. We found that D5- related peptide inhibits HK-biotin cellular uptake. Confocal microscopy showed that HK colocalizes with heparan sulfate proteoglycan (HSPG) at the cell surface. When biotin-HK is incubated with rabbit aorta endothelial cells (RAECs) and CHO-K1 cells, it is internalized into acidic intracellular vesicles, whereas when incubated with CHO-745 cells, which express reduced levels of glycosaminoglycans, HK is not internalized. To further verify the hypothesis that HSPG-dependent mechanisms are involved in HK uptake and proteolytic processing in lysosomes, we tested chloroquine, which blocks Alexa 488- HK colocalization with Lyso Tracker in acidic endosomal vesicles. The process of HK internalization was blocked by low temperatures, methyl-beta-cyclodextrin, FCCP and 2-deoxy-D-glucose, implying that HK uptake into acidic vesicles is energy-dependent and most likely involves binding to HSPG structures localized in cholesterol-rich domains present in the plasma membrane. Kinin generation at the cell surface was much higher in tumorigenic cells (CHO-K1) when compared to endothelial cells (RAECs). The present data indicate that the process of HK endocytosis involving HSPG is a novel additional mechanism which may control kinin generation at the cell surface.

Interaction of proteinase inhibitors with phospholipid vesicles is modulated by pH

“…The reported findings also provide clear evidence for the occurrence of pH-induced electrostatic interactions with phospholipid vesicles and point to Bauhinia inhibitors as valuable tools for the understanding of diverse mechanisms, which correlate their biological properties of inhibiting proteinase activity. For example, since BbCI is known to inhibit specifically a lysosomal cathepsin L enzyme at pH 4.5, our results suggest that, under acid pH conditions, the inhibitors may interact with cell membrane and are still able to inhibit uncontrolled proteolysis (showed by its functional and structural stability), as it occurs with kininogens, the major plasma cystatin-like inhibitor, which bind to the cell membrane, mediated by different structures, internalizes and act on cysteine cathepsin [41][42][43]. These findings may help to clarify the action of similar plant inhibitors on biological models, and pH-induced conformational changes, to understand some functional aspects of the protein in vivo, particularly under pathological conditions, connected with local fluctuations of pH.…”

Modulation of the Plasma Kallikrein-Kinin System Proteins Performed by Heparan Sulfate Proteoglycans

“…The current research group is the first to show that the interaction of H-kininogen and PK on the cell surface mediated by HSPGs results in endocytosis (Melo et al, 2009; Veronez et al, 2014; Damasceno et al, 2015; Figure 1). H-kininogen interacts with cellular sites in either RAECs or epithelial CHO cells, wild type CHO-K1 cells and CHO-745 cells, mutant cells deficient in xylosyltransferase and as consequence is involved in GAG biosynthesis.…”

“…At neutral pH and after H-kininogen assembly on the cell surface, kinin generation was much higher in CHO-K1 cells, and tumorigenic cells, when compared with RAECs, as expected, as either serine or cysteine proteases are involved in kinin release. In CHO-745 cells, only serine protease activity is detected consistently with the regulation of cysteine proteases by GAGs/PGs in H-kininogen processing and kinin release (Melo et al, 2009; Damasceno et al, 2015). The lysate fraction of CHO-K1 cells possesses kininogenase activity at pH7.4 but the total cleavage of intact H-kininogen is more effective at pH 5.5 after a 4 h incubation.…”

“…The interaction parameters of H-kininogen with RAECs and CHO cells are strongly dependent on temperature and the interaction between H-kininogen and cells was not totally reversible at 37°C; the interaction with CHO cells is related to vital processes also influenced by ATP synthesis; the H-kininogen interaction in CHO cells is independent of cycloheximide, indicating that new protein synthesis does not influence H-kininogen binding. GAGs are not the main cellular receptors; however, GAGs modulate the cellular receptors of H-kininogen in CHO cells (Melo et al, 2009). …”

“…The endocytosis process of H-kininogenin CHO-K1 cells is disrupted by pretreatment of the cells using chloroquine, an alkalinizing agent of acid endosomes; cholesterol depletion by methyl-β-cyclodextrin also inhibits endocytosis; the endocytosis of H-kininogen is independent of transferrin endocytosis, strongly indicating that the endocytosis of H-kininogen is not dependent on clathrin. The membrane lipid raft domains/caveolae mediate the endocytotic process of H-kininogen, as the colocalization of H-kininogen with caveolin-1 has been demonstrated (Melo et al, 2009; Damasceno et al, 2015). …”