Differential regulation of intestinal amino-oligopeptidase gene ...

Differential regulation of intestinal amino-oligopeptidase gene expression in neonatal and adult rats MITCHELL

L. COHEN,

NILDA

A. SANTIAGO,

JIA-SHI

ZHU,

AND

GARY

M. GRAY

Department of Medicine, Division of Gastroenterology, and The Digestive Disease Center, Stanford University School of Medicine, Stanford, California 94305

COHEN, MITCHELL L., NILDA A. SANTIAGO, JIA-SHI ZHU, AND GARY M. GRAY. Differential regulation of intestinal aminooligopeptidase gene expression in neonatal and adult rats. Am.

J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G866-G871, 1991.-Intestinal amino-oligopeptidase(AOP) is an essential brush-border hydrolytic enzyme required for the surface digestion of nutrient oligopeptidesproducedfrom luminal pancreatic protease action on dietary protein. There is an abrupt rise in AOP catalytic activity during postnatal rat development, but the mechanism has not been defined. AOP expression was examined in rats 11 to 60 days of ageby measurementof AOP mRNA, catalytic activity, and total AOP protein (by quantitative rocket immunoassay).Specific catalytic activity began increasingat 18 days, achieveda maximum by 22 days (+125% over 11 days), and remained stable thereafter. A l.l-kb AOP cDNA, generatedby the polymerasechain reaction and usedto quantify specific mRNA, identified a single 3.8-kb speciesat all ageson Northern blots. The abundance of @-actin mRNA, which increasedslightly (+40%), and 7s RNA, which did not change,wasalsomeasuredasdevelopmentalcontrols. The AOP mRNA-to-7S RNA ratio increaseddramatically (+410%) between 11 and 60 days of age.A comparableinitial rise in AOP activity (+130%) and in its mRNA (+170%) was observed between 11 and 22 days, followed by a divergence of the two curves, with a marked relative excessof mRNA comparedwith catalytic activity in the 60-day-old adult. The ratio of catalytically active to total immunoreactive AOP protein was higher in 60-day-old adults compared with both ll- to X-day-old preweaned(65% of 60-day value) and 22- to 30-day-old postweaned (61% of 60-day value) animals. These results suggest there is differential control of AOP gene expressionwhereby the initial rise in catalytic activity during the maturational period, regulatedpretranslationally, is followed in the adult by a stable catalytic activity maintained via translational and/or posttranslational mechanisms. aminopeptidaseN; membraneglycoproteins; mRNA; ,&actin; 7s RNA; jejunum; ontogeny

LIGOPEPTIDASE~ (AOP), which cleaves amino acids from the NH2-terminus of oligopeptides of two to six residues (X5), is a large glycoprotein hydrolase present in high concentrations in the intestinal brush border of mammals (11, 31). This microvillar enzyme plays an essential role in the surface digestion of nutrient oligopeptides produced by luminal pancreatic proteases in preparation for intestinal transport of the final dipeptide and amino acid products (29). Its synthesis is regulated AMINO-•

’ Also called aminopeptidase dase M, L-leucyl+naphthylamide dase. G866

N, oligoaminopeptidase, hydrolase, and neutral 0193-1857/91

$1.50

aminopeptiaminopeptiCopyright

by intraluminal peptide nutrients (24). During rat development, there are dramatic alterations in small intestinal structure and function, including microvillar enzyme expression, that occur primarily at the time of weaning during the third postnatal week (10). Dietary changes do not appear to play a causal role in these maturational events, but rather hormonal influences, particularly glucocorticoids and thyroxine, are important mediators of intestinal ontogeny (10). AOP specific catalytic activity rises dramatically during this developmental period, with the increase primarily localized to the jejunum (2, 19). The mechanisms underlying this marked induction of AOP expression have not yet been defined, however. In intestinal explants, Danielsen et al. (5) noted a marked increase in the rate of synthesis of AOP in the adult compared with the fetal pig, despite comparable amounts of AOP mRNA, which were determined indirectly by translation in a cell-free system. While these results suggest that regulation of AOP synthesis during development may be translationally controlled, the use of a specific cDNA probe is necessary to directly quantify specific mRNA levels as a function of maturation. We have generated a specific cDNA for rat intestinal AOP by the polymerase chain reaction (PCR) and have used it to examine the expression of mRNA coding for this essential brush-border hydrolase during postnatal development. This has enabled us to directly correlate specific mRNA during maturation with catalytically active and total (immunoreactive) AOP. EXPERIMENTAL

PROCEDURES

MateriaZs. Guanidinium thiocyanate and hydrochloride were purchased from Fluka; oligo( dT) -cellulose from Collaborative Research; GeneAmp DNA amplification reagent kit from Perkin Elmer Cetus; reverse transcriptase, human placental RNase inhibitor, T4 polynucleotide kinase, restriction enzymes, DNA polymerase Klenow fragment, and radioisotopes from Amersham; GeneClean kit from Bio 101; pBluescriptI1 and calf intestinal alkaline phosphatase from Stratagene; T4 DNA ligase from New England Biolabs; Sequenase 2.0 sequencing kit from US Biochemical; Nitroplus nitrocellulose membranes from Micron Separations; formamide from Kodak; formaldehyde, dextran sulfate, salmon sperm DNA, leucyl-P-naphthylamide, and Triton X-100 from Sigma; protein assay reagent and Tris/Tricine buffer (immunoelectrophoresis Tricine buffer IV) from

0 1991 the American

Physiological

Society

DEVELOPMENTAL

EXPRESSION

Bio-Rad; agarose (SeaKem ME) and Gel-Bond from FMC. Oligonucleotide primers were synthesized by Genetics Designs. Adult rat kidney mRNA for use in the polymerase chain reaction was from ClonTech Laboratories. Animals. All rats were purchased from Simonsen Laboratories in Gilroy, CA. Jejunal mRNA for use in the polymerase chain reaction was isolated from the pooled tissue of three adult male Sprague-Dawley rats. For developmental studies, all rats were of the Wistar strain except in experiments measuring total AOP immunoprotein, when Sprague-Dawley rats were used. PCR amplification of rat intestinal AOP cDNA. Based on the sequence of rat kidney aminopeptidase N (30), an upstream oligonucleotide primer, 1325+ (Y-CTGAATGATGTGTACCGTGTG-3’), and two downstream primers, 2022- (Y-GCTAGGTTGAAGGAGTCGTG3’) and 2410- (Y-GCAGTAGACAGTAGACCGAAG3’) were synthesized. PCR amplifications were carried out using either adult Sprague-Dawley rat intestinal mRNA, prepared as described below, or kidney mRNA (ClonTech Laboratories) as the template. The GeneAmp DNA amplification reagent kit was used and standard lOO-~1 reactions were set up according to the manufacturer’s instructions, supplemented with 20 U of reverse transcriptase and 20-40 U of human placental RNase inhibitor. Incubation was carried out in an Eppendorf DNA Microcycler at 42°C for 60 min for first-strand cDNA synthesis followed by 25 cycles of PCR consisting of chain denaturation at 94°C for 1 min (2 min for first cycle), annealing of primers at 55°C for 1 min, and primer extension at 72°C for 3-5 min (3 min for 9 cycles, 4 min for 8 cycles, 5 min for 7 cycles, and a final 15-min cycle to complete polymerization of all strands). Aliquots of the individual PCR products were then electrophoresed on a 1% agarose gel (26). Cloning and sequencing. A l.l-kb cDNA amplified by PCR from rat intestinal mRNA was purified by agarose gel electrophoresis by use of GeneClean, 5’-phosphorylated with 20 U of T4 polynucleotide kinase (26), and then ligated via T4 DNA ligase into the pBluescriptI1 plasmid vector that had been cut with Sma I and dephosphorylated with calf intestinal alkaline phosphatase. This cDNA insert was then sequenced to verify that it represented a partial-length cDNA for AOP. Partial sequencing of both strands of the double-stranded DNA insert by initial alkaline denaturation followed by dideoxy chain termination reactions was carried out using the Sequenase 2.0 kit. DNA denaturation was carried out at 95°C for 2 min, followed by annealing of sequencing primers at room temperature for 15 min; the labeling reactions were performed in an ice-slush bath for 2.5 min and terminated at 55°C for 4 min. Samples were then run on a 5% polyacrylamide gel using a 38 X 50 cm BioRad apparatus (Sequi-Gen). Tissue preparation. For correlative studies of AOP catalytic activity and mRNA content, three Wistar rat litters allowed free access to maternal milk and standard rat food (Wayne MRH 22/5 Rodent Blox) were used. Three 60-day-old male Wistar rats (260-280 g), which served as adult control animals, were also allowed free access to rat food, and each was randomly paired with one of the rat litters. Each set of animals was then studied

OF

AMINO-OLIGOPEPTIDASE

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separately, yielding three sets of data. At 11 A.M. on postnatal days 11, 15, 18, 22, and 30, two rat pups from each litter were killed; the jejunum was removed, cleared of any residual contents with a flush of cold 154 mM NaCl, and divided alternately into 2-cm and l-cm segments for total RNA isolation and enzymatic assay, respectively; and the tissue from the two littermates was then pooled. The jejunum from each adult rat was processed in an identical fashion. Total kidney RNA was also isolated from the pooled kidney tissue of the three adult rats. For studies of the total AOP immunoprotein expressed during intestinal maturation, three Sprague-Dawley rat litters randomly paired with 60-day-old adult males (260280 g) were used. Newborns were allowed to suckle ad libitum and standard rat food was available continuously. Rat pups were killed during postnatal days 11-15 (preweaned group) and 22-30 (postweaned group), the jejunum was removed and cleared of residual contents, and the particulate fraction was then prepared, solubilized, and assayed for AOP activity (see below). The tissue from adult animals was processed identically. Each solubilized particulate sample was prepared from one or two rats. Solubilized brush-border protein for use as an AOP standard in these experiments was prepared from the combined mucosal scrapings of eight 60-day-old male Sprague-Dawley rats and assayed for AOP activity (see below). AOP and protein assays. Leucyl-P-naphthylamide hydrolase activity, a specific assay for AOP, was assayed as previously described (31) and expressed in units (U = pmol/min). Total protein was determined by the BioRad assay. RNA isolation. Total RNA was isolated by the lowtemperature guanidinium thiocyanate method (9). The quantification of individual total RNA samples by absorbance at 260 nm and their integrity was confirmed by ethidium bromide staining after fractionation on denaturing 2.2 M formaldehyde, 1% agarose gels (26). mRNA was isolated by oligo(dT)-cellulose chromatography (26). Northern blot hybridization analysis. Samples of total cellular RNA (7.5 pg/lane) were electrophoresed on denaturing 2.2 M formaldehyde, 1% agarose gels as described above and then transferred to nitrocellulose membranes (26). Blots were probed with the l.l-kb AOP cDNA excised from pBluescript with BamH I and Pst I and labeled with [a-32P]dCTP (3,000 Ci/mmol) to a specific activity of lo8 to 10’ cpm/pg DNA by the random primer method with DNA polymerase Klenow fragment (6). Prehybridization was performed in 50% (vol/vol) formamide, 5 x SSC (1 x SSC = 150 mM NaCl, 15 mM sodium citrate), 5 x Denhardt’s solution, 25 mM potassium phosphate buffer (pH 7.4), and 50 pg/ml salmon sperm DNA. Hybridization was carried out for 16-18 h at 42°C in prehybridization solution to which 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), and the radiolabeled probe (5 x lo5 cpm/ml) were added. Blots were washed at room temperature twice in 1 X SSC plus 0.1% SDS for 15 min and then twice in 0.25 X SSC plus 0.1% SDS for 15 min. The blots were then exposed to Kodak XAR-2 film at -70°C using a single intensifying screen. The AOP mRNA in each tissue RNA preparation was quantified by cutting the bands representing the

G868

DEVELOPMENTAL

EXPRESSION

AOP mRNA hybridized to its specific radiolabeled cDNA probe from the blots and determining the radioactivity present by liquid scintillation counting. Each filter was then sequentially rehybridized with the developmental controls, first at 42°C with the 1.2-kb BgZ I fragment of the rat ,&actin cDNA (21) and then at 37°C with the 190-bp mouse 7s RNA cDNA (3), and quantitation of mRNA was carried out similarly. Preparation and solubilization of membranes. Brushborder membranes precipitated by 10 mM CaClz treatment and 50,000 g X 20 min particulate fractions were prepared as described previously (1). Membranes were solubilized by sonication in 1% Triton X-100 (1). Rocket immunoelectrophoresis. Quantitative rocket immunoelectrophoresis was performed essentially as described by Laurel1 (16), except that the 1% agarose gels were made in 27 mM Tris/Tricine buffer containing the previously characterized monospecific anti-AOP antiserum (8) and layered onto a Gel-Bond backing. For the study samples, 0.5 mU (20 ~1) of AOP catalytic activity was applied to the wells. Aliquots of the solubilized brush borders from 60-day-old adult rats, prepared identically, served as the AOP standard. Gels were run at 65 V at 4°C for 12 h on a horizontal immunoelectrophoresis unit (LKB). After electrophoresis, gels were washed serially in 1 M (30 min), 0.5 M (1 h), and 0.2 M (1 h) NaCl and distilled water (1 h X 2), and were then stained in 0.01% Coomassie Brilliant Blue and dried overnight at 37°C. Analysis of data. Data were expressed as means + SE. Statistical analyses were performed by one-way analysis of variance, the two-tailed Student’s t test, or both. RESULTS

PCR amplification and sequencing of rat intestinal AOP cDNA. The results of amplification of rat intestinal and

kidney mRNA using oligonucleotide primers based on the sequence of rat kidney aminopeptidase N (30) are shown in Fig. 1. Indistinguishable products, amplified with intestinal or kidney mRNA, were of the expected 0.7- and l.l-kb lengths based on the distance between the primers. This was highly suggestive that the amplified products from intestinal mRNA represented the

1.49 l.l-

-1.1

kb

+0.7

kb

0.90.6-

FIG. 1. Agarose gel electrophoresis of polymerase chain reaction (PCR)-amplified products. Intestinal or kidney mRNA (0.5 pg) from adult male Sprague-Dawley rats was reverse transcribed and the DNA template amplified with specific upstream (+) and downstream (-) oligonucleotide primers based on the sequence of rat kidney aminopeptidase N (30). Ten percent of each reaction product was then run on a 1% agarose gel and visualized by ethidium bromide staining.

OF AMINO-OLIGOPEPTIDASE

aminopeptidase N or AOP cDNA and that the enzyme was identical in both rat kidney and intestine. Further evidence to support this was provided by a Southern blot analysis identifying the l.l-kb PCR product using the 2022- rat kidney aminopeptidase N PCR primer as an internal probe and by the presence of an EcoR I site -0.5-kb from the 5’ end of the cloned l.l-kb cDNA, the location of the restriction site in kidney aminopeptidase N (30) (data not shown). Finally, sequencing of the first 193 bp at the 5’ end and 259 bp at the 3’ end of the cDNA revealed it to have 100% identity with kidney aminopeptidase N and established conclusively that it represented the rat intestinal aminopeptidase N or AOP cDNA. AOP catalytic activity

during postnatal development.

Figure 2 shows the pattern of AOP catalytic activity in the rat jejunum during postnatal development. Through the end of the third week of life, total jejunal AOP activity demonstrated a steady increase that exceeded the corresponding rise in total protein concentration. As a result, AOP specific catalytic activity (per mg protein) rose 125% by 22 days of age (Fig. 2, right ordinate). This initial marked rise was then followed by a small but insignificant decrease in AOP specific activity at 30 and 60 days. Overall, the doubling of activity apparent at 22 days was maintained in older animals. AOP mRNA levels during postnatal development. The abundance of AOP mRNA in the jejunal RNA samples from an individual representative set of rats, determined by Northern blot analysis, is shown in the autoradiogram in Fig. 3. The AOP cDNA probe hybridized to a single 3.8-kb mRNA species in both jejunal and kidney specimens, the known size for the rat kidney aminopeptidase N mRNA (30), and there was no significant degradation of RNA. Notably, the radioactive signal increased appreciably during the developmental period from 11 to 60 days of age. In marked contrast, levels of the developmental controls, @actin mRNA, and particularly 7s 35t

1.6 1

30

Age (days) 2. Pattern of amino-oligopeptidase (AOP) catalytic activity during postnatal development. AOP catalytic activity and total protein content were measured on jejunal homogenates, as described in EXPERIMENTAL PROCEDURES, and expressed per rat jejunum. AOP activity at each age was also divided by corresponding protein concentration (specific activity), and the ratio expressed as a fraction of the go-dayold adult values. There were 3 sets of data at each time point, except at 11 days of age when only 2 pairs of animals could be studied because insufficient RNA was isolated from the third litter. Comparison of the specific activity (means + SE) in rats from 11 to 30 days of age was performed by one-way analysis of variance (P C 0.04). FIG.

DEVELOPMENTAL k&t

lld

Jejunum 18d 226

154

306

6Od

EXPRESSION

Adult Kidney

7.5\’ 4.4-

\

cAOP(3.8

kb)

2.4C C7S

p-Actin RNA

FIG. 3. Representative Northern blot of intestinal RNA during development. Total cellular RNA (7.5 pg/lane) isolated from jejunum of ll-, 15-, 18-, 22-, 30-, and 60-day-old (adult) rats and kidneys of 60day-old (adult) rats was electrophoresed on a denaturing 2.2 M formaldehyde, 1% agarose gel, transferred to a nitrocellulose membrane (26), and then probed with the l.l-kb AOP cDNA labeled with [o~-~‘P] dCTP by the random primer method (6). After washing of the membrane and exposure to Kodak XAR-2 film, the blot was sequentially rehybridized with the 1.2-kb rat @-actin cDNA (21) and the 190-bp mouse 7s RNA cDNA (3) as developmental controls (insets). Exposure times for the autoradiograms: 19 h, AOP; 3.5 h, P-actin; and 8 h, 7s RNA.

1. AOP and P-actin mRNA and 7s RNA during rat intestinal development

TABLE

Age, days

AOP mRNA

11 15 18

1.0+0.06 1.320.5 2.OzkO.3 2.7kO.5 3.4k0.6 4.5kO.3

22 30 60

@-Actin

mRNA

-7s RNA

0.9kO.2

1.OkO.l l.lkO.2

l.lkO.2

1.0+0.04

1.1kO.l

1.0+0.02 1.1+0.1 0.9+0.1

l.OkO.2

1.4kO.2 1.4kO.02

ANOVA P < 0.002 P = 0.2 P = 0.8 Values are means f SE. The relative abundance of each RNA species was quantified by cutting the bands from the Northern blots and determining the radioactivity by liquid scintillation counting. Mean values at each age are expressed relative to 11 days, which is arbitrarily normalized to a mean value of 1.0. Animal characteristics are as described in the legend to Fig. 2. Values in each group were compared by one-way analysis of variance (ANOVA). 6.0


G869

RNA were relatively constant throughout this period. Average values for the relative abundance of AOP and @-actin mRNA and 7s RNA for the three sets of animals were derived by directly measuring the radioactivity in excised bands from the Northern blots. As shown in Table 1, there was a marked change in AOP mRNA levels, amounting to an increase of 2.4-fold by 30 days of age and 3.5fold by 60 days of age. During this period, there was a much smaller mean increase (40%, but not a significant change; P = 0.2) in the level of P-actin mRNA and no change in the abundance of 7s RNA (P = 0.8). To normalize the data from the individual blots and to correct for the small variation in the loading of RNA in different lanes, we expressed the level of AOP mRNA as a ratio to the highly stable 7s RNA (Fig. 4). Compared with the ll-day preweaning values, AOP mRNA levels increased progressively during development, achieving a maximum (+410%) in the 60-day-old adult. Direct comparison of the magnitude of change in AOP specific catalytic activity (relative to total protein) with mRNA levels (relative to 7s RNA) in the developing rat jejunum revealed a comparable dramatic increase in both between 11 and 22 days of age (Fig. 4). However, after weaning, a further marked increase in the relative mRNA level was associated with a stable specific catalytic activity. As a result, a marked excess of mRNA nearly triple the relative catalytic activity was seen in the go-day-old adult animal. Total immunoreactive AOP protein during intestinal maturation. To investigate whether the discrepancy be-

tween the AOP catalytic activity and mRNA level in the 60-day-old adult (Fig. 4) was due to the appearance of a catalytically inactive form of the enzyme, we measured total AOP protein by rocket immunoelectrophoresis. Figure 5 displays graphically the ratio of catalytically active to total immunoreactive AOP protein in ll- to E-dayold preweaned, 22- to 30-day-old postweaned, and 60day-old adult rats. Rather than displaying a reduction in the activity of the AOP, there was a much higher ratio of active to total AOP protein in the adult (arbitrarily

-I-

n q

Catalytic Act&y mRNA

Age (days) FIG. 4. Comparison of AOP catalytic activity with mRNA levels during postnatal development. Mean values for the AOP catalytic activity (expressed per mg protein; see Fig. 2) and the AOP mRNA (expressed relative to the 7s RNA control; see Table 1) are normalized to 11 days, which is arbitrarily given a mean value of 1.0. The specific catalytic activity and relative mRNA level at each age, directly compared by Student’s t test, differed only at 60 days (*P < 0.006). Also one-way analysis of variance revealed an increase in AOP mRNA after 22 days (P < 0.05), despite no significant change in catalytic activity over the same period (P = 0.7).

Pre-W Post-W AM ’ FIG. 5. Ratio of catalytically active to immunoreactive AOP during intestinal maturation. Relative amount of total AOP protein was determined by quantitative rocket immunoelectrophoresis in ll- to 15day-old preweaned (Pre-W), 22- to 30-day-old postweaned (Post-W), and 60-day-old adult male (AM) rats. At least 3 determinations were made in each group. Mean values for the ratio of catalytically active to total immunoreactive AOP were expressed relative to AM rats (normalized to a mean of 1.0). Overall comparison of the 3 groups was performed by one-way analysis of variance (P < 0.04). By Student’s t test, AM rats differed significantly from both Pre-W (*P < 0.05) and Post-W (*P < 0.05) rats.

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EXPRESSION

defined as 1.0) compared with preweaned (0.65) and postweaned (0.61) animals. Thus, because a smaller fraction of the AOP was enzymatically active in the developing rat, the total AOP protein expressed was relatively higher than estimated by catalytic activity during maturation. DISCUSSION

Molecular species of AOP mRNA in rat jejunum. This paper represents the first comprehensive study of the developmental mRNA expression of an intestinal brushborder digestive peptidase. Using a radiolabeled Ll- kb AOP cDNA (Fig. 3), we have identified by Northern analysis a single 3.8-kb mRNA species in the developing rat jejunum identical in size to the major species previously identified in the adult rat kidney (30). Whereas a second mRNA species of 3.4 kb was found in the kidney, our results are compatible with the findings of a single AOP mRNA species in the rabbit, pig, and human jejunum (22). Mechanism of induction in AOP catalytic activity during weaning. Similar to previous studies (2,19), we found

the dramatic increase in AOP specific catalytic capacity during postnatal development to take place primarily during the third postnatal week (Fig. 2), the period when marked structural and functional changes are known to occur in the small intestine associated with the weaning process (10). Because a comparable rise was observed in both the specific catalytic activity and the relative mRNA level (Fig. 4), this change appears to result predominantly from regulation of the enzyme by a pretranslational mechanism at the level of gene transcription or by a reduction in the rate of intracellular mRNA degradation. These findings differ dramatically from those reported by Danielsen et al. (5) in pig intestinal explants in which the rate of synthesis of fetal AOP was only 3% of the adult rate, despite comparable amounts of AOP mRNA in fetal and adult intestine as determined indirectly by translation in a cell-free system and identical primary translation products of AOP, as estimated from SDS-polyacrylamide gel electrophoresis. These findings might reflect either technical difficulties associated with measuring mRNA levels indirectly by translation in a ceil-free system or possibly an interspecies difference. Our findings based on direct measurement of mRNA levels by a specific cDNA probe strongly support the conclusion that the dramatic increase in AOP expression occurs predominantly by a pretranslational rather than by a translational or posttranslational mechanism. Regulation of intestinal AOP gene expression in the adult mammal. After the third postnatal week, other

regulatory mechanisms also appear to be involved in the modulation of AOP gene expression. During this period, despite maintenance of a stable specific catalytic activity, the AOP mRNA level continued to increase to maximum values in the 60-day-old adult rat. Direct comparison of the catalytic activity with the mRNA level confirmed this observation (Fig. 4). In addition, the measurement of total immunoreactive AOP demonstrated that the discrepancy between the AOP catalytic activity and mRNA level in 60.day-old adult rats could not be exnlained bv the appearance of a catal-ytically inactive form

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AMINO-OLIGOPEPTIDASE

of the AOP. Instead, there was a significantly higher active fraction of AOP in the adult animal (Fig. 5). This indicates rats 11-30 days of age have an appreciable inactive fraction (35-40%) of AOP. Thus, when an estimate is made of the ratio of AOP mRNA to total AOP protein in 60-day-old adult rats, there is an even greater excess of mRNA relative to total AOP protein (ratio of 4.6) than to AOP catalytic activity (ratio of 3.0). The increase in the active fraction of AOP in the adult animal presumably acts to offset the appreciable drop in total AOP protein. These findings indicate that regulation of AOP gene expression after the developmental period changes appreciably, occurring by mechanisms operating predominantly at the translational and possibly posttranslational levels. The possible mechanisms include inhibition of mRNA translation, structural or conformational changes in the glycoprotein making it more active catalytically, and increased degradation of the AOP. The plasma membranes of most mammalian cells possess a wide array of integral glycoproteins that play vital biological roles in functions ranging from digestion in the case of intestinal hydrolases, to growth regulation, antigenicity, and membrane transport of electrolytes and solutes, and many have also been shown to act as receptors for bacterial toxins, viruses, and a variety of hormones (13). Yet little is known about the detailed mechanisms regulating their gene expression during maturation. There are several examples of a proportional change in the mRNA levels and expression of membrane glycoproteins, including the digestive hydrolases, sucrase-isomaltase (17, 27, 28) and dipeptidyl peptidase IV (12), and membrane transport proteins such as the glucose transporter (25) and the Na+-K+-ATPase (18). However, there are very few examples of control at the translational or posttranslational level. The abrupt decline in intestinal lactase activity during the weaning period is not accompanied by a proportional decline in its mRNA (7), and the decline in catalytic activity appears to be due to synthesis of an inactive form of the enzyme (20, 23) and to an increase in degradation after membrane insertion (4). For sucrase (17), an initial parallel rise in both its mRNA and specific catalytic activity during weaning is followed by an apparent drop in enzymatic activity and a suggestion of stable mRNA levels in 36day-old animals, indicating subsequent translational or posttranslational regulation. It is not possible, however, to draw any firm comparisons from that study because older animals were not examined (17). The estrogendependent rise in prolactin receptor levels during female sexual maturation exhibits an initial comparable rise in both hepatic prolactin receptor numbers and mRNA that is followed subsequent to 40 days of age by a continuing increase in receptor expression in the face of stable mRNA levels (14). This form of dual regulation is strikingly different from that observed for AOP, not only because it is associated with female sexual development rather than with weaning and maturation but also because the changes are opposite in direction to those observed for AOP, there being a further incremental increase in mRNA out of proportion to the expression of this digestive peptidase in the adult animal. Indeed, we

DEVELOPMENTAL

EXPRESSION


G871

could find no other similar example of such a clear-cut biphasic mechanism of developmental regulation. The reason for this persistent relative increase in AOP mRNA in the adult is not apparent, but the regulation of gene expression after weaning is clearly very complex and occurs at mu1 .tiple levels. Therefore, other mechanisms operating at the translational and/or posttranslational levels appear to be important for AOP regulation after weaning. The exact mechanisms whereby pretrancontrol is slational, translational, and posttranslational achieved at different stages during postnata .1 development will require further study.

acterization of aminopeptidase N from the human small intestine. Biochem. Res. 2: 517-526, 1981. 12. HONG, W. J., J. K. PETELL, D. SWANK, J. SANFORD, D. C. HIXSON, AND D. DOYLE. Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels. Exp. Cell Res. 182: 256-

The authors thank Dr. Murray Korc (UC Irvine) for generously providing the mouse 7s RNA cDNA. We also thank Drs. Wing K. Kam (UC San Francisco), Christian Oste (Cetus Corp., Emoryville, CA), Stanley Falkow, Anson W. Lowe, Larry A. Scheving, and Kenneth T. Denich for their advice and valuable suggestions during the course of this work, and Dr. Byron W. Brown, Jr., for his guidance with the statistical analysis. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-15802 and DK-38707. M. L. Cohen was supported by NIDDK Training Grant DK-07056, Individual National Research Service Award DK-08267, and Clinical Investigator Award DK-01948. Address for reprint requests: G. M. Gray, Div. of Gastroenterology, Room SO69, Stanford Univ. School of Medicine, Stanford, CA 94305.

52, 1966. 17. LEEPER,

266,1989. 13. JOHNSON,

L. R. (Editor). Physiology of the Gastrointestinal Tract (2nd ed.). New York: Raven, 1987. 14. JOLICOEUR, C., J. M. BOUTIN, H. OKAMURA, S. RAGUET, J. DJIANE, AND P. A. KELLY. Multiple regulation of prolactin receptor gene expression in rat liver. Mol. Endocrinol. 3: 895-900, 1989. 15. KANIA, R. K., N. A. SANTIAGO, AND G. M. GRAY. Intestinal surface amino-oligopeptidases. II. Substrate kinetics and topography of the active site. J. Biol. Chem. 252: 4929-4934, 1977. 16. LAURELL, C. B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. AnaZ. Biochem. 15: 45-

18.

19.

20.

21.

Received 19 December 1990; accepted in final form 18 June 1991. 22.

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