Horseradish peroxidase binding to intestinal brush-border membranes ofCyprinus carpio. Identification of a putative receptor

Abstract

Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush-border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (K(d) = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin-type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3-kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization.

Undetectable apolipoproteinA-Igene expression suggests an unusual mechanism of dietary lipid mobilisation in the intestine ofCyprinus carpio

“…Internalisation of a wide variety of intact proteins across the intestinal epithelium has been demonstrated for several teleost fish (Moriyama et al, 1990;Hertz et al, 1992;Vera et al, 1992). In the carp, a specific receptor protein for the endocytosis tracer horseradish peroxidase has been identified by ligand blotting (Amthauer et al, 2000;Concha et al, 2002) but no specific protein could be identified as a putative receptor for apoA-I, utilising a similar approach (data not shown).…”

“…ApoA-I binding assays Brush border membrane vesicles (BBMV) were isolated from carp intestinal mucosa and characterised before their use in the binding assays as described by Amthauer et al (2000). Prior to the binding assays the test tubes were blocked by incubation with 1·mg·ml -1 BSA for 1·h at 0°C to avoid unspecific binding of 125 I-apoA-I to the test tube wall.…”

“…Radiolabelled proteins were detected by autoradiography. For western blot analysis the proteins separated by SDS-PAGE were transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA) using a semi-dry blotter unit (Amthauer et al, 2000). Membranes were blocked with 5% (w/v) non-fat dry milk in PBS/Tween-20 (0.1% v/v).…”

Teleost intestinal immunology

“…The receptor mediated uptake of HRP results in a very fast peak of HRP in the blood of carp [29] and trout [30] 30 min after oral administration, while the appearance of ferritin or LPS inside macrophages is more a process of 4e8 h [7,21]. The HRP receptor is presently characterized, but the biological reason for this receptor mediated uptake of enzymes still not [31]. In contrast to mammalian M cells enterocytes of the second segment have functional endolysosomal organels and are not well able to phagocytose inactivated bacteria [32].…”

Gut Immune System and the Implications of Oral-Administered Immunoprophylaxis in Finfish Aquaculture

“…Fluid phase uptake of soluble antigens such as ferritin has been proven to take place via non-specific pinocytosis in grass carp ( Ctenopharyngodon idella ) and rainbow trout ( 38 , 70 – 72 ). For small solid particles (<0.5 μm), as exemplified by HRP, solid-phase uptake by receptor-mediated endocytosis has been reported in the fish gut ( 18 , 73 ). For larger particulate antigens, uptake can occur via phagocytosis, where the antigens are surrounded and internalized by the protrusion of cell membrane forming phagosomes ( 18 , 74 ).…”