key: cord-0905548-lcvbaguj
authors: Bolte, Gabriele; Knauss, Margarete; Metzdorf, Irene; Stern, Martin
title: Postnatal Maturation of Rat Small Intestinal Brush Border Membranes Correlates with Increase in Food Protein Binding Capacity
date: 1998
journal: Dig Dis Sci
DOI: 10.1023/a:1018844608861
sha: 13d7be55943b3e57e2ddecbbd8fc9f73589fd5dd
doc_id: 905548
cord_uid: lcvbaguj

To investigate maturational changes of membranefood protein binding capacity, we studied bindingcharacteristics of brush border membranes isolated fromsmall intestines of newborn and adult rats. Binding of biotinylated gliadin peptides, cow's milkproteins (α-casein, β-lactoglobulin,α-lactalbumin, bovine serum albumin) and lectinswas assessed by a sensitive chemiluminescence blotassay. We found specific food protein binding with regardto saturation and inhibition. Maximal binding of mostfood proteins and several lectins to brush bordermembranes of newborn and adult rats was comparable, whereas binding of β-lactoglobulin wassubstantially less. Common or adjoining binding sitesfor the different food proteins tested were indicated bycorresponding membrane protein binding patterns and by inhibition properties of unrelated proteins.Compared to newborns, adult membrane vesicles as well asisolated membrane proteins showed higher bindingcapacities. Thus postnatal maturation of smallintestinal brush border membranes correlated withincreased food protein binding capacity.

In addition to their barrie r function and participation in dige stive , absorptive , and se cretory proce sse s, small inte stinal e nte rocyte s were propose d to contribute to mucosal immune re actions by pre senting antige ns coming from the gut lume n (1, 2) . Binding of fore ign prote ins to the apical brush borde r membrane (BBM) was reporte d to facilitate uptake and to in¯uence intrace llular proce ssing (3± 5). Interactions of luminal molecules with the BBM also could re sult in signal transduction and cellular re sponse s (6, 7) .

Rats are a suitable mode l for studying deve lopm ental change s in BBM archite cture and enterocytic functions, because profound change s occur during post-natal maturation. At the time of weaning, the shift from milk-base d to carbohydrate -rich fee ding is accompanie d by change s of BBM, in e nzyme activitie s, prote in and lipid composition, glycosylation, and membrane¯uidity (6, 8 ± 10) . Furthe rmore, the ne wborn rat mucosal barrie r appe ars to be more leaky, as indicate d by highe r rate s of endocytosis (11, 12) . Enhance d inte raction of prote ins with BBM is suppose d to contribute to the increased uptake see n in ne wborn rats (10) .

Con¯icting results were obtaine d e arlie r in our laboratory using diffe rent approache s to study intestinal food prote in binding in vitro. In e xpe rime nts using whole gut sacs, we found a maturational increase in binding and uptake of b -lactoglobulin and bovine se rum albumin whe n comparing immature 18-day-old rats with mature adult rats (13) . In contrast, a maturational decrease in the binding capaci-ties of isolate d BBM ve sicles from ne wborn to adult age was de monstrate d for a -lactalbumin, b -lactoglobulin, bovine se rum albumin, and the gliadin pe ptide B3142 (14) . O ur aims, therefore , we re to clarify whether e nhance d BBM food prote in binding occurs before weaning and to characte rize membrane compone nts that inte ract with food prote ins. Food prote ins of clinical importance (gliadin pe ptide s, cow's milk prote ins, ovalbumin) and le ctins (controls for spe ci® city of binding and for de te ction of de velopmental change s in membrane glycosylation) were use d. Anim als. Adult Sprague-Dawley rats, weighing 200 ± 300 g, and timed-pregnant rats were obtained from Charles-River Wiga (Sulzfeld, Ge rmany). They we re kept on a glute n-fre e and cow's milk protein-poor rat chow (diet no. C1078, Altromin, Lage, Ge rmany). Newborn rats were allowed to suckle fre ely and we re killed at the age of 24 hr. Adult rats were fasted one day prior to the study.

Brush Border Mem bran e Vesicles. For isolation of brush border membrane vesicles, whole small intestines of newborn rats and proximal halves of adult small intestines we re removed and washed with cold physiological saline. Starting with homogenates of neonatal intestines or scrapings of adult intestinal mucosa, BBM ve sicles were obtained by a modi® ed Ca 2 1 -precipitation technique as described before (16) . For e ach preparation, small intestines of several newborn rats or mucosa scrapings of two adult rats we re pooled. Activities of the BBM enzymes lactase , sucrase, alkaline phosphatase , and total maltase activity we re dete rmined according to Dahlqvist (17) and Bowers and McComb (18) .

One unit was de® ned as 1 m mol substrate hydrolyzed per minute under the e xperimental conditions. Speci® c e nzyme activities were expressed as units per milligram of protein.

Protein concentrations we re measured using bovine serum albumin as standard (19) .

Electron Microscop y. BBM ve sicles we re ® xed in 2% (v/v) glutaraldehyde , 100 mM cacodylate buffe r, pH 7.4, for 1 hr at 4°C and collecte d by centrifugation (30 min at 30,000g). Pe llets were washed three times with cacodylate buffe r, pellete d, and post® xe d in 1% (v/v) osmium te troxide in cacodylate buffe r for 1.5 hr at room tempe rature. Afte r washing, the me mbranes we re dehydrate d ste pwise in e thanol and tre ate d with saturated uranyl acetate for contrast enhanceme nt. Ultrathin sections were prepared from me mbrane vesicled e mbedded in Araldite, stained with lead citrate, and analyze d with a Z eiss EM10 e lectron microscope.

Biotin ylation . Food proteins were labeled with biotin, using biotinamidocaproate-N-hydroxysuccinimide ester for cow's milk proteins and biotinamidocaproate-N-hydroxysulfosuccinimide ester for gliadin peptides as described previously (15) .

Gel Electrop horesis. Sodium dodecyl sulfate ± polyacrylamide ge l electrophoresis (SDS-PAGE ) was performed using the Mini-Protean II e lectrophoresis system (Bio-Rad, Munich, Germany) according to Laemmli (20) under nonreducing conditions. Western Blot. Electrophoretic transfe r of proteins from polyacrylamide ge ls to nitrocellulose shee ts was performed by tank blotting as described before (21) , using the Mini Trans-Blot electrophoresis transfe r cell from Bio-Rad. To enhance transfe r, SDS was adde d in a ® nal concentration of 0.05% (w/v) to the electrode buffer.

Ch em ilum in escence Binding Assay. Intact BBM ve sicles or e lectrophoretically separate d membrane proteins were immobilized on nitrocellulose sheets by the dot blot (22) or we ste rn blot technique. Free binding sites of nitrocellulose we re blocked with 2% (v/v) Twe e n 20 in phosphatebuffe red saline (PBS), pH 7.4. To diminish nonspeci® c adhe rence, 0.05% (v/v) Tween 20 in PBS was used in each washing or incubation step. Afte r incubation with biotinylated proteins overnight at 4°C, bound probes we re detecte d using peroxidase-conjugated streptavidin and ECL blotting dete ction reage nts. Chemilumine scence signals we re qua ntitate d d e nsitom e trica lly with the vide odensitomete r syste m Biopro® l (LTF Labortechnik, Wasserburg, Ge rmany), using the program Bio-1D ve rsion 5.08 (V ilber Lourmat Biotechnology, Marne La Valle e, France). Binding intensity was e xpressed as densitometric units per microgram BBM protein in relation to biotinylated standard proteins (Bio-Rad), which we re applied directly to nitrocellulose. Saturation and inhibition of protein binding we re assessed as criteria for binding speci® city. Inhibition of protein binding was measured by addition of nonlabeled proteins to solutions of biotinylated probes. Change s in binding we re calculated in relation to binding of biotinylated proteins alone. Maximal binding intensities were calculated from results of saturation e xperiments.

Statistical An alysis. All data are expressed as mean 6 standard deviation (SD). Student's t te st was used for statistical analysis of diffe rences of BBM binding capacities betwe en newborns and adults.

Ch aracterization of BBM Vesicles. BBM vesicle s were isolate d from rat small inte stines, forming close d vesicle s of varying size s. Cytoske leton prote ins of the brush borde r appe are d as e le ctron-de nse mate rial inside the vesicle s ( Figure 1 ). Enrichm ent of BBM marke r enzyme s indicate d suf® cie nt purity of membrane preparations (Table 1) MAA binding was not saturate d in the concentration range te ste d ( Figure 3 ). Food prote in binding to adult BBM re ache d 18.6 to 27.4 de nsitom e tric units/m g BBM prote in e xce pt BLG, which was signi® cantly less bound to BBM ( Table 2) . Adult BBM bound signi® cantly more PT-GLI, CAS, BLG, ALA, BSA, and O V A than ne wborn membrane s. Lectin binding re¯ected known change s in membrane glycosylation during postnatal maturation. According to the shift from sialylation to fucosylation, ne wborn BBM bound more PNA, MAA, and SNA, but less UEA I ( Table 2 ).

In h ibition of Bin din g. Be sides saturation, spe ci® city of food prote in binding to BBM was studie d by inhibition e xpe riments. Binding of PT-GLI, CAS, BLG, and ALA was inhibite d by a 10 3 -fold e xcess of nonlabe le d prote in. Howe ver, the obse rved aggre gation of BSA and O VA, de ® ne d as the ability of the biotinylate d prote in to bind to the immobilize d unmarke d counte rpart, was not compatible with this kind of e xpe rime nt (Figure 4) . O nly in the case of weak binding due to low conce ntrations of biotinylate d prote ins was a 10 3 -fold exce ss of nonlabe le d prote in suf® cient to achie ve 100% inhibition (data not shown) . With concentrations above 0.01 m g/ml, assay sensitivity did not allow 100% inhibition of speci® c le ctin binding, too, using e ithe r nonlabe le d lectins or monosacchari des (Figure 4 ). Food Protein Bin din g to Isolated Mem bran e Protein s. Binding characte ristics of isolate d membrane prote ins were furthe r analyze d in weste rn blots to de ® ne maturational diffe rences. In accordance with results obtaine d with whole BBM in dot blots, isolate d membrane prote ins of adult rats showe d highe r binding capacitie s for PT-GLI, ALA, BLG, and O V A and more binding sites than ne wborn rats ( Figure 5 ). This maturational diffe rence was also se e n with BSA and CAS (de nsitom etric pro® les not shown) . Common or at least adjoining binding sites for food prote ins were de te cted on newborn membrane prote ins of 124 ± 131 kDa, 117± 120 kDa, and 66 kDa. Adult membrane prote ins of 128 ± 138 kDa, 115± 120 kDa, 103± 105 kDa, 90 ± 92 kDa, 85± 86 kDa, 65± 67 kDa, and 45± 46 kDa share d food prote in binding abilitie s. To e xclude that the se maturational diffe rences were due to methodical errors, e g, failure to isolate ne wborn BBM prote ins, lectin binding was studie d in western blots, too. Newborn membrane prote ins had more binding site s and e nhance d binding capacity for SNA, PNA, and MAA than adults, whe re as UEA I binding was lowe r in ne wborns compare d to adults (data not shown) .

Inhibition e xpe rime nts with unre late d prote ins indicate d common binding of food prote ins be cause cow' s milk prote ins, e spe cially CAS, were able to re duce BBM binding of an unre late d prote in, whe reas O V A interfe red to a lower e xte nt with cow' s milk prote in and gliadin peptide binding (Table 3) . It was not possible to discriminate whe the r inte rfere nce was due to prote in interactions in solution, to nonspe ci® c membrane binding, or to the pre sence of common binding site s. On the othe r hand, cow' s milk prote ins and O V A did not inte rfe re with le ctin± BBM interactions and did not disturb the che milumine scence reaction. Addition of a 10 4 -fold e xce ss of nonlabe led food prote ins to solutions of biotinylate d lectins at a conce ntration of 0.1 m g/ml did not re sult in signi® cant change s of le ctin binding. Due to its strong adhe rence to BBM, only CAS was able to re duce le ctin binding by 19.4 ± 37.7% .

Diffe rent approache s have be en re porte d to study BBM± prote in interactions and to characte rize deve lopme ntal change s in BBM structure . Be side histochemical studie s (23, 24) and utilization of isolate d enterocyte s (25) and of gut sacs (3, 13, 26, 27) , BBM vesicle s have bee n use d. As the smalle st unit for studying membrane structure and function (28) , BBM vesicle s allow description of e nte rocytic binding structure s in detail, at the same time reducing the complexity of the inve stigate d syste m. Binding characte ristics of rat BBM ve sicle s for food prote ins and le ctins have bee n asse ssed in centrifugation and ® ltration binding assays (14, 16, 29 ± 33) . A conside rable drawback of the se methods was the ir low sensitivity in de tecting weak food prote in binding.

To improve se nsitivity, we use d a chemilumine scence binding assay in this study. Food prote in binding to BBM of ne wborn and adult rats was found to be spe ci® c with re gard to saturation and inhibition. Ove rall food prote in binding capacity of BBM was comparable to le ctins, except for BLG. Saturation of food prote in binding was only re ached at 10-to 100fold highe r molar conce ntrations compare d to le ctins and CAS. This differe nce betwe en food prote ins and le ctins is in accordance with former studie s (16, 33) . In contrast, the former centrifugation binding assay did not show any saturation and inhibition of food prote in binding to BBM vesicle s (13, 14, 16) . In our opinion, results of nonspe ci® c food prote in binding to BBM were obscure d by methodological dif® cultie s in the centrifugation binding assay, which did not allow washing of ligand± BBM comple xe s without concurrent loss of binding signals.

While Colye r et al (25) reporte d up to 50% inhibition of gliadin pe ptide binding to isolate d e nte rocyte s, Farre Castany et al (34) demonstrate d nonspe ci® c binding of a pe ptic± tryptic dige st of gliadin to the colon carcinom a e pithe lial cell line HT-29. The authors studie d bindin g prope rtie s of ovalbum inblocke d and glutaralde hyde -® xed HT-29 cells, which might be an e xplanation for the obse rve d concentration-de pe nde nt, but nonspe ci® c, gliadin binding. Nevertheless, gliadin is able to associate with many prote ins, mainly by hydrophobic interactions (35) . To exclude weak hydrophobic inte ractions be twee n food prote ins and BBM, we studie d binding phe nomena in pre sence of the de te rge nt Twee n 20.

Experiments with BBM vesicle s in dot blots gave a marke d incre ase in food prote in binding capacitie s from ne wborn to adult rats. This ® nding contradicts pre vious re sults of a maturational de crease of food prote in binding to rat BBM (14, 16) and might be due to the more sensitive and spe ci® c binding assay use d in this study. Howe ver, our ® ndings are in accordance with data on increased binding and uptake of BLG and BSA obtaine d in gut sacs of adult rats compare d to juve nile rats (13) .

For measure ment of strong and highly speci® c le ctin binding to BBM, the binding assay system was not found as critical. The same maturational change s in BBM glycosylation were obse rve d using e ithe r the chemilumine scence binding assay in this study or othe r methods as de scribe d e lse where (29, 30, 32) . Neve rthe le ss, minor differe nce s indicate d enhance d sensitivity of the chemilum inescence assay. Binding of SNA to adult BBM and of UEA I to ne wborn membrane prote ins was measurable , in contrast to the earlie r re sults of Taatje s and Roth (24) and Le noir et al (36) , which were base d on a diffe rent methodology.

The maturational decrease in the lipid/prote in ratio of BBM (10, membrane prote in pro® les in Figure 5 ) is accompanie d by an increase in food prote in binding to isolate d BBM prote ins. In western blots, membrane prote ins of adult rats had more binding site s and highe r binding capacitie s than BBM prote ins of ne wborn rats. Although membrane prote ins are denature d during SDS-PAGE , the ir binding prope rtie s are not necessarily de stroye d, as has be en shown for seve ral re ceptor± ligand inte ractions in weste rn blots (37, 38) .

O ur data of multiple binding site s do not support the hypothe sis of distinct re ceptors for food prote ins comparable to the e nzyme aminope ptidase N as a receptor for coronavirus (39) . Moreover, prote in± oligosaccharide inte ractions le ading to membrane attachme nt could be e xclude d be cause food prote in binding patte rns did not show any similarity to le ctin patte rns in western blots. Lectin-like binding characte ristics of BSA and ALA binding to rat BBM vesicle s and of gliadin to model glycoprote ins were rule d out earlie r by othe r inve stigators (27, 40) . In our opinion, prote in± prote in inte ractions are responsible for food prote in attachme nt to BBM. This kind of interaction was obviously not in¯uence d by the diffe re nt composition and physicoche mical characte ristics of the investigate d cow's milk prote ins, gliadin pe ptide s, and ovalbumin. O n the contrary, common or at le ast adjoining binding site s e xist for the diffe re nt food prote ins, as indicate d by western blot ® ndings. Inhibition by unre late d food prote ins supports this possibility. Control e xpe riments could exclude artifacts cause d by biotinylation of food prote ins. V e sicle de struction leading to re le ase of brush borde r cytoske leton prote ins could possibly in¯uence binding characte ristics re corde d in We ste rn blots. For example , actin as a major compone nt of adult brush borde r might have caused the positive signals in the are a of 45 kDa. There fore furthe r e xpe rime nts have to be conducte d to ide ntify structure s in membrane binding and their orientation towards the intestinal lume n in vivo.

In conclusion, contrary to e arlie r re ports, BBM food prote in binding was found increased during ente rocytic maturation of rat small intestine. It has to be ke pt in mind that any experimental de sign using isolate d BBM vesicle s in vitro lacks important parts of the mucosal barrie r (41) . Diffe rences in prote in re sistance to inte stinal hydrolysis (gliadin, BLG in contrast to CAS) (42) in¯ue nce the ir acce ss to small intestinal e pithe lial cells in vivo. For e valuation of conseque nces of change s in food prote in binding capacity cause d by alte rations in BBM composition, it should be inve stigate d whe the r proce sse s of uptake and intrace llular processing or signal transduction occur. It is well-known that endocytosis is stimulate d by prior membrane adsorption (43, 44) . Moreover, diffe rence s of intrace llular proce ssing after uptake of membranebound prote ins or pinocytosis of¯uid-phase molecule s have bee n re porte d for small intestinal e pithelial cells (4, 5, 45) . O ur results clearly indicate that BBM composition in¯ue nces its capacity for speci® c food prote in binding. In this re gard, studie s of enterocytic membrane alte rations possibly involve d in the de velopm ent of food-se nsitive e nte ropathie s would be worthwhile .

Pre se ntation of peptides and proteins by intestinal epithelial cells

Murine ente rocyte s can prese nt soluble antigen to spe ci® c class II-re stricted CD4 1 T cells

Intestinal uptake of retinol: Enhance ment by bovine milk b -lactoglobulin

Me mbrane -bound and¯uid-phase macromolec ules e nter se parate pre lysosomal compartmen ts in absorptive cells of suckling rat ileum

Rode wald R: Evidence for the sorting of endocytic vesicle contents during the receptor-me diated transport of IgG across the newborn rat intestine

Intestinal brush borde r me mbrane function

In vivo re sponses of rat intestinal epithelium to intraluminal dietary lectins

Postnatal changes in biosynthesis of microvillus me mbrane glycans of rat small intestine: I. Evidence of a de velopme ntal shift of te rminal sialylation to fucosylation

Mechanism of maturational de cline of rat intestinal lactase ± phlorizin hydrolase

Structure± function relationships in intestinal brush border me mbrane s

The uptake of¯uoresce in-conjugated dextran 70,000 by the small intestinal e pithelium of the young rat and pig in re lation to macrom ole cular transm ission into blood

Lactobacillu s casei strain GG reve rse s increase d intestinal perme ability induced by cow milk in suckling rats

Food proteins and gut mucosal barrie r. II. Differential interaction of cow' s milk proteins with the mucous coat and the surface me mbrane of adult and immature rat jejunum

Food proteins and maturation of small intestinal microvillus membrane s (MVM). III. Food protein binding and MV M proteins in rats from newborn to young adult age

Peptic± tryptic dige sts of gliadin: Contaminating trypsin but not pepsin interfere s with gastrointestinal binding charact e ristics

Food proteins and maturation of small intestinal microvillus me mbrane s (MV M). I. Binding characteristics of cow's milk proteins and concanavalin A to MVM from ne wborn and adult rats

Assay of intestinal disaccharidase s

A continuous spe ctrophotome tric me thod for me asuring the activity of serum alkaline phosphatase

Protein me asure ment with the Folin phe nol reagent

Cleavage of structural proteins during the assembly of the head of bacteriophage T4

Electrophore tic transfe r of proteins from polyacrylamide ge ls to nitrocellulose she ets: Procedure and some applications

A dot-immunobinding assay for monoclonal and other antibodies

Pe tell JK: Re activity of gliadin and lectins with ce liac intestinal mucosa

Se lective loss of sialic acid from rat small intestinal e pithelial ce lls during postnatal deve lopment: De monstration with lectin-gold techniques

Gliadin binding to rat and human ente rocyte s

Uptake of polypeptide fragments by rat intestine in vitro and in vivo

Effect of surface desialylation on intestinal uptake of proteins in suckling rats

Me thodological guide for studying epithelial transport with isolated membrane ve sicle s. In Cell Membrane Transport. Expe rime ntal Approache s and Methodologie s. DL Yudilevich, R De ves

Postnatal change s in lectin binding to microvillus me mbrane s from rat intestine

Deve lopment of gastrointestinal surface. V III. Lectin identi® cation of carbohydrate differe nces

Banwe ll JG: Binding of isolectins from red kidney bean (Phaseolus vulgaris) to puri® e d rat brush-border me mbrane s

Ne onatal and adult patterns of lectin binding to rat small intestinal microvillus me mbrane s

Crypt± villus differe ntiation re¯e cte d by lectin and protein binding to rat small intestinal brush border me mbrane s

Tlaskalova -Hoge nova H: Binding of gliadin to lymphoblastoid, myeloid and epithelial ce ll lines

Inve stigation of gliadin binding to differe nt se lected proteins using a biotin± stre ptavidin syste m

De velopm e ntal ch an ge s in inte stinal glyco syl ation: Nu tritionde pendent multi-factor re gulation of the fucosylation pathway at weaning time

Immunoautoradiographic detection of e pide rmal growth factor receptors after e lectrophore tic transfer from gels to diazo-pape r

V isualization of lactotransferrin brush-border re ceptors by ligand-blotting

Human aminopeptidase N is a receptor for human coronavirus 229E

Koe ttgen E: Studies on the aetiology of coeliac disease: No e vide nce for lectin-like components in wheat glute n

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Transport of me mbrane -bound macromol e cule s by M ce lls in follicleassociate d epithelium of rabbit Pe ye r's patch

E ndocytosis and the recycling of plasma membrane

E ndocytosis in absorptive cells of cultured human small-intestinal tissue: Horseradish peroxidase , lactope roxidase, and ferritin as markers

The authors thank Dr. A. O sman and Dr. T. Mothes, Institute of Clinical Chemistry and Pathological Biochemistry, Unive rsity of Leipzig, for providing gliadin peptides. The help of Dr. H. Wolburg, Institute of Pathology, University of Tuebingen, in e lectron microscopy studies and of P.-M. We ber in preparing photographs is grate fully acknowledge d.