Direct inhibitory effect of rotavirus NSP4(114-135) peptide on the Na(+)-D-glucose symporter of rabbit intestinal brush border membrane

Abstract

The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport of D-glucose and of L-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na(+)-D-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na(+)-L-leucine symport about equally and partially via a different transport mechanism, in that Na(+) behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.

FIG. 1

Kinetic effects of NSP4-related peptides on d-glucose uptake by intestinal BBM vesicles from 4-week-old rabbits. Initial rates of d-glucose uptake, expressed as the uncorrected, absolute uptake rates, are plotted as a function of substrate concentration in either the absence (■) or presence of 1 mM NV(464-483) (□), mNSP4(131K) (○), and NSP4(114-135) (●). Solid lines represent theoretical fits computed by using the kinetic parameters listed in Table 1. However, because the control, NV(464-483), and mNSP4(131K) results were all statistically indistinguishable, they have been pooled and are shown as a single curve corresponding to the overall fit for 4-week-old rabbits in Table 1. The insert shows the results obtained by using only the lowest [d-glucose] range used, 0.1 to 10 mM.

FIG. 2

Dose-dependent effects of the NSP4(114-135) and NV(464-483) peptides on the initial rate of d-glucose uptake by jejunal BBM vesicles from 7-week-old rabbits. Fixed amounts of vesicles were mixed with variable amounts of peptide in the appropriate volume of membrane buffer to give the indicated (final) peptide concentrations. These mixtures were either used directly (cis peptide conditions) or subjected first to two cycles of freezing and thawing to permit equilibration of the extra- and intravesicular contents (cis-plus-trans conditions), as described elsewhere (24). After incubation for 5 min at 35°C, aliquots were used to measure the initial rate of 0.1 mM d-glucose uptake under standard conditions. Because the results at all given cis and cis-plus-trans peptide concentrations were statistically indistinguishable, they have been pooled and fitted again to obtain relevant, overall fits. These are illustrated by the same symbol, ● for NSP4(114-135) and □ for NV(464-483). Results are shown as absolute substrate uptake rates ± SD. The solid lines represent theoretical fits of either set of data to equation 2, computed by using the kinetic parameters listed in Table 2.

FIG. 3

Dose-dependent effects of the NSP4(114-135) and NV(464-483) peptides on the initial rate of l-leucine uptake by jejunal BBM vesicles from 7-week-old rabbits. Details are as for Fig. 2, except for the use of l-leucine as the substrate and peptides only in the cis side. Symbols: ●, NSP4(114-135); □, NV(464-483). Because the two peptides gave statistically indistinguishable results, the two sets of data have been pooled to obtain the overall fit in Table 2 (heavy middle line).

FIG. 4

Effect of the NSP4(114-135) peptide on the kinetics of l-leucine uptake, according to the full, nonobligatory three-site symport model. Experimental l-leucine data (taken from Fig. 3) were fitted by nonlinear regression analysis to equation 3. The theoretical fits thus computed show the overall result to be the sum (ct) of two distinct hyperbolas: one that is Michaelian (concave; c1) and another that is convex (c2).