1 Department of Medicine and 3 Digestive Disease Center, Stanford University School of Medicine, Stanford, California 94305; and 2 Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo, Ohio 43614
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ABSTRACT |
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The structure of aminooligopeptidase (AOP), an intestinal brush-border digestive hydrolase, is abnormal in human diabetes and in the congenitally diabetic BioBreed Wistar (BBd) rat. Its assembly in the BBd rat was examined. After normal initial synthesis and assembly of immature AOP precursor (AOPi) with high-mannose N-linked chains in the endoplasmic reticulum (ER), processing of N-linked glycans in Golgi yielded a smaller than normal mature AOP precursor (AOPm) with persistence of some high-mannose N-linked chains. Deglycosylation analyses suggested that the mass difference could be attributed to a lower mass of N-linked with unaltered O-linked glycans in AOPm of the diabetic rat. Intrajejunal pulse-chase experiments revealed that the conversion of AOPi to AOPm occurred at 30 min of chase in normal rats but at 60-90 min in diabetic rats, reflecting delay in ER-to-Golgi transport or a slower processing of high-mannose chains. Once maximal transport to Golgi was achieved, the residence time in Golgi was shortened in diabetes. This altered processing of the precursor accounted for the altered structure of AOP in diabetes.
aminopeptidase N; kinetics; N-linked carbohydrates
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INTRODUCTION |
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AMINOOLIGOPEPTIDASE (AOP), commonly known as aminopeptidase N, resides in the brush-border surface membrane of epithelial cells of the renal proximal tubules (31, 32) and is most prominent at the intestinal enterocytic surface (15). Analogous aminopeptidases are also expressed variably in other tissues such as splenocytes, hepatocytes, salivary glands, and the genital tract (31). Although the physiological significance of AOP in most organs is uncertain, AOP plays a pivotal role in the small intestine in protein assimilation by acting at the lumen-enterocyte interface to cleave sequentially the NH2-terminal amino acid residues from nutrient oligopeptides having two to six amino acid residues (17). The released amino acids are then transported by the enterocyte.
In vivo pulse-chase experiments with radioactive amino acid precursors in the intact rat showed initial glycosylation of AOP with high-mannose N-linked chains in association with the endoplasmic reticulum (ER) to yield maximal incorporation in an immature AOP precursor (AOPi) after only 15 min of chase (2). Subsequent posttranslational processing of N-linked chains to complex forms and probable addition of O-linked chains occur after its transport to the Golgi compartment, producing a mature AOP precursor (AOPm) by 45 min. AOPm is then transported to the brush-border surface without additional modification.
Since its discovery in 1974, the spontaneously diabetic BioBreed Wistar
(BBd) rat has been considered an excellent experimental model for the study of type 1 diabetes mellitus in humans because of
many common histopathological and clinical features (20). Previously, we demonstrated (23) an altered structure of
newly synthesized and steady-state brush-border AOP in the
BBd rat manifested by a 5-kDa reduction in mass. Similarly,
the structure of another enterocyte digestive enzyme,
sucrase--dextrinase, was also altered in the BBd rat
(22), suggesting that altered glycoprotein structure is
common in congenital diabetes. The alteration is independent of
hyperglycemia or the degree of severity of diabetes and reverts to
normal over ~3-wk treatment of the diabetic animal with insulin (22, 23). Deglycosylation of brush-border AOP and
sucrase-
-dextrinase revealed normal apoprotein products in the
BBd rat, suggesting that the mass change of AOP and
sucrase-
-dextrinase in congenital diabetes is caused by altered
carbohydrate chains (22, 23).
In the present studies, we have examined the intracellular synthesis and assembly of intestinal AOP in the intact diabetic BBd rat. We have identified abnormal posttranslational processing of its N-linked carbohydrate chains that results in a mass change and altered kinetics of its membrane-associated assembly and trafficking through the ER and Golgi compartments. Additionally, we have examined the structure of AOP in human type 2 diabetes. Similar to findings in the congenitally diabetic BBd rat (23), we observed a smaller than normal AOP in human subjects. This suggests that altered AOP structure is common in diabetes.
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MATERIALS AND METHODS |
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Materials. All chemicals and reagents are Baker analyzed reagents except where otherwise indicated.
Experimental animals.
Male BioBreed BBd diabetic and normal (Wist) Wistar rats
were selectively bred in the Animal Resources Division, Health
Protection Branch, Health and Welfare, Ottawa, ON, Canada, and sent to
the animal facility at Stanford University 1-10 days after the
onset of diabetes. All rats were housed in a room lighted from 6:00 AM
to 6:00 PM in cages equipped with filters (Lab Care caging system;
Research Equipment, Byran, TX) and fed regular laboratory rat chow ad
libitum. The BBd rats developed spontaneous diabetes at
60-90 days of age and weighed 250-350 g. After preliminary experiments established that Wist rats showed no structural change in
AOP from 60 to 120 days of age (23), they were age- and
weight matched with BBd rats as controls. BBd
rats required insulin for survival at the onset of diabetes. They were
kept on a daily dose of neutral protamine Hagedorn (NPH) insulin
(U-100; Lilly) injected subcutaneously at 4:00 PM. Withdrawal
of insulin for 24 h resulted in a hyperglycemic state (serum
glucose level 300 mg/dl). One day before the experiment, rats were
fasted overnight and kept solely on drinking water. At 7:00 AM the
following day, rats were either injected intraperitoneally with
[3H]leucine or pulse-chased intraluminally with
[35S]methionine (see Intraluminal labeling of
proximal jejunum of rats). During the experiment whole
blood glucose was assayed by a glucose analyzer (Boehringer Mannheim,
Indianapolis, IN), and urinary and blood ketone and glucose were
checked by the Ketostix and Dipstick methods (Roche Diagnostics),
respectively. Serum was kept at
20°C for later determination of
D-(
)-
-hydroxybutyrate, a ketoacidosis marker
(33), and glucose (Beckman Glucose Analyzer-2).
Human tissues.
Jejunal segments (25-60 g) of normal and type 2 diabetic
African-American men and women were obtained during surgery or
immediately after death from the National Disease Research Interchange
(Philadelphia, PA). After removal, tissues were quickly frozen and kept
at 70°C for 2-20 mo. The ages of the donors varied between 50 and 80 yr.
Intraluminal labeling of proximal jejunum of rats.
A proximal jejunal segment (15- to 20-cm length) prepared in rats
anesthetized with pentobarbital sodium as described previously (1) was preperfused for 1 h with 0.9% NaCl (37°C)
to remove pancreatic proteases from the lumen. It was then pulsed with
2 mCi of L-[35S]methionine (1,200 Ci/mmol;
Amersham) for 5 min and chased with 1 mM L-methionine for
15-180 min. After its removal, the jejunal segment was flushed
with 0.9% NaCl and 1 mM dithiothreitol (4°C) and the mucosa was
scraped off with a glass microscopic slide and homogenized in 10 ml/g
tissue of buffer A (5 mM histidine, pH 7.4 with imidazole, 5 mM EDTA) including protease inhibitors (chymostatin, antipain
hydrochloride, leupeptin, and ovomucoid trypsin inhibitor, final
concentration 25 µg/µl; Sigma, St. Louis, MO). The
homogenate was then subjected to serial differential centrifugation,
and the organelles were separated on a final 25-60% sorbitol
gradient as described previously (1) with sucrase as a
specific marker for brush border and -mannosidase II for the Golgi
membranes (24). The ER and Golgi membranes comigrated on
this gradient and were pooled together as ER-Golgi fractions (ERG)
(1).
Immunoprecipitation and electrophoresis.
ER-Golgi and brush-border fractions were solubilized in 0.5% Triton
X-100 and assayed for AOP activity as described previously (15). AOP was then specifically immunoprecipitated with
monospecific polyclonal AOP antiserum (1), washed, heated
at 100°C for 5 min in 200 µl of SDS buffer (63 mM
Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 10 mM
dithiothreitol, and 1 mM EDTA), and analyzed by 7% SDS-electrophoresis
and autoradiography (Kodak X-OMAT films at 70°C) as described
previously (2). The ratio of the distance from the origin
to the AOP band relative to the distance from the origin to the gel
front (Rf) was measured independently by four observers,
and the molecular mass was then estimated by plots of log
Rf vs. molecular mass of Bio-Rad standards. In some
experiments, the AOP immune pellet was treated with enzymatic probes
before electrophoresis or the bands were excised from the dried gels and the radioactivity quantified after solubilization in
Solulyte-Lipofluor-water (1:10:0.2). In other experiments, the
autoradiogram was scanned on an imaging densitometer (Eagle Eye II,
Stratagene) and the proteins were quantitated with the NIH Image
(version 1.60) Macintosh software program.
Treatment by endo--N-acetylglucosaminidase H.
The AOP immunoprecipitate (see Immunoprecipitation and
electrophoresis) was taken up and boiled in 100 µl of 150 mM
sodium citrate (pH 5.5), 2% SDS for 2 min, and 1 mU of
endo-
-N-acetylglucosaminidase H (Endo H; Boehringer
Mannheim) per 75 mU of AOP was then added to the supernatant (15,000 g, 5 min) as described previously (10). Briefly, Endo H (10 mU) was added to the recovered supernatant, and the
digestion mixture was placed at 37°C for 1 h with a thin overlay
of toluene. Control samples were treated identically except that
incubation was in buffer without Endo H. After addition of albumin (15 µl of 10 mg/ml) to enhance recovery of the protein pellet, the Endo
H-treated samples were precipitated with 10% trichloroacetic acid,
washed with cold acetone (
20°C), and analyzed by SDS-PAGE.
Treatment of AOP by N-glycosidase F.
The AOP immunoprecipitate was washed and taken up in 100 µl
of 0.2 M sodium phosphate buffer (pH 8.6), 2% SDS, 2% mercaptoethanol at 100°C for 2 min. The supernatant (15,000 g, 5 min) was
then collected and treated with N-glycopeptidase F
(PNGase F; Boehringer Mannheim) as described previously
(10). Briefly, the SDS supernatant was brought up to 900 µl with the sodium phosphate buffer, 1% NP-40, and 0.5%
mercaptoethanol. Five units of PNGase F in 100 µl of the same
buffer were then added, and the mixture was covered with a thin overlay
of toluene at 37°C for 18 h. The reaction was stopped by placing
the tubes on ice. Albumin (150 µg) was added as the protein carrier,
and trichloroacetic acid (final concentration 10%) was added to
precipitate AOP protein. The mixture was then rotated on a wheel at
4°C for 1-2 h. The protein pellet (15,000 g, 5 min)
was washed with acetone (20°C), taken up in 200 µl of SDS buffer,
and applied to 7% SDS-acrylamide gel electrophoresis as described above.
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RESULTS |
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Altered N-linked chains in brush-border AOP.
Wist and BBd rats were injected intraperitoneally
with 4 mCi of [3H]leucine and killed 4 h
later. Brush-border AOP was solubilized, immunoprecipitated, and
incubated with either Endo H or PNGase F or in buffer alone without
either enzymatic probe before electrophoresis (Fig.
1). Untreated AOP from BBd
migrated as a 135-kDa structure, 5 kDa smaller than normal (140 kDa)
(Fig. 1; Rf of 0.388 in Wist vs. 0.407 in
BBd). AOP from normal rats displayed no significant mass change when treated with Endo H (Fig. 1), indicating essentially complete processing of N-linked carbohydrates to complex structures during maturation of the precursor in the Golgi membranes. In contrast,
Endo H treatment of AOP from the brush-border membranes (BBM) of
BBd rats produced a heterogeneous band ranging between 130 and 135 kDa (Fig. 1), suggesting persistence of some Endo H-sensitive
high-mannose glycans or hybrid N-linked chains in brush-border AOP of
the BBd rat. Removal of N-linked carbohydrates by
PNGase F produced a single broad band that migrated as a similar structure of ~120 kDa in both Wist and BBd rats (Fig. 1;
Rf of 0.455 in Wist and 0.459 in BBd rats),
suggesting that there was no additional posttranslational structural
alteration of AOP in the BBd rat such as modification of
its O-linked chains in the Golgi compartment.
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Normal protein synthesis and glycosylation with high-mannose
N-linked carbohydrates of AOP in the BBd rat.
The finding of altered N-linked glycosylation (with no
accompanying alteration in O-linked structures) of brush-border AOP in
the BBd rat (Fig. 1) prompted the analysis of the
intracellular synthesis and processing of the AOP precursor. For this
purpose, rats were administered 2 mCi of [35S]methionine
intraluminally into a jejunal segment for 5 min and chased with 1 mM
methionine for 30 and 60 min (normal) and 60 and 90 min (diabetic) to
achieve maximal incorporation into the ERG (see MATERIALS AND
METHODS). AOP (40-55 mU) from solubilized ERG was recovered,
treated with Endo H, and analyzed by electrophoresis and
autoradiography (Fig. 2). The
AOPi was indistinguishable in normal and diabetic
BBd rats [Fig. 2A, Endo H lane at 30 min (normal) vs. Fig. 2B, Endo H
lane at 60 min (diabetic)].
Similarly, removal of high-mannose N-linked chains by Endo H produced a
more rapidly migrating apoprotein [AOPi(e)] that
displayed an identical pattern for both rat groups [Fig.
2A, Endo H+ lane at 30 min (normal) vs. Fig. 2B,
Endo H+ lane at 60 min (diabetic)]. The Rf of
AOPi and AOPi(e) were indistinguishable in
normal and diabetic rats and amounted to 0.422-0.425 and
0.451-0.454, respectively, when AOP extracted from ER-Golgi
fractions after 15 min of chase was applied on the same gel (data not
shown). This suggested that protein synthesis and initial
cotranslational assembly of high-mannose carbohydrates proceeded
normally in the BBd rat.
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Altered intracellular maturation of N-linked carbohydrate chains of
AOP in the BBd rat.
Wist and BBd rats were pulse-chased for 15, 60, and 150 min
as described in MATERIALS AND METHODS.
[35S]methionine-labeled AOP was then isolated from ERG
and BBM fractions, treated with PNGase F and analyzed by
SDS-electrophoresis and autoradiography (Fig.
3). AOPi at 15 min appeared
initially in the ERG as a major band in both normal and diabetic rats
(Fig. 3) and was subsequently converted to a larger mature
AOPm species that was eventually transported to the BBM in
both diabetic and normal animals (Fig. 3). Removal of N-linked chains
of AOPi at 15 min of chase by PNGase F yielded an
apoprotein product [AOPi(pf)] indistinguishable in the normal and diabetic rat (Fig. 3), supporting our observation of normal protein synthesis and initial carbohydrate assembly in the ER of the diabetic rat (Fig. 2). Determining the molecular masses of AOPi and AOPm by log-linear
plots of Rf vs. mass before and after removal of N-linked
high-mannose and complex N-linked chains by PNGase F (Fig. 3), as
represented in Table 1, revealed that the
change in AOPm mass is caused by alteration of
N-linked chains during maturation. For instance, there was no detectable difference in the mass of AOPi for the
BBd and Wist rats (125 kDa), and removal of N-linked chains
by PNGase F produced an apparently identical apoprotein product of
~110 kDa in each case (Table 1). Thus the mass of high-mannose
N-linked chains of AOPi appeared to be indistinguishable in
the BBd and Wist rats and amounted to ~15 kDa (Table 1).
However, the conversion of AOPi to AOPm
involved the addition of ~15 kDa for the Wist rat (Table 1; 125140
kDa) and only 10 kDa of mass for the BBd rat (Table 1;
125
135 kDa) during the maturation of the precursor in the Golgi
membranes. Removal of N-linked carbohydrates by PNGase F from
AOPm yielded a similar residual mass (120 kDa) in both Wist
and BBd rats [AOPm(pf); Fig. 3 and Table 1].
Because PNGase F removes both high-mannose and complex N-linked chains,
the decrease in the apparent molecular mass of AOPm after
PNGase F treatment from 140 to 120 kDa in the Wist rat and from 135 to
120 kDa in the BBd rat accounts for a total mass of ~20
kDa for N-linked chains in normal and ~15 kDa in BBd rats
(Table 1). The apparent mass of O-linked chains, added during the
conversion of AOPi to AOPm in the Golgi
compartment, can be estimated from the difference between
AOPm(pf) (N-linked chains removed; putative
O-linked chains attached) and AOPi(pf) (the
completely deglycosylated apoprotein). Notably, this mass difference in
the molecular mass of AOPi(pf) and AOPm(pf) is
~10 kDa in both Wist and BBd rats; hence the mass of
their O-linked chains (possibly also including other
posttranslationally added structures) appears to be indistinguishable
from normal in congenital diabetes (Table 1). These data confirm those
of Fig. 1.
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Delayed maturation of AOPi in the ERG in vivo. As noted above, the 15-min chase experiments suggested normal synthesis and assembly of the AOPi precursor in the ER of the diabetic animal (Fig. 3B vs. Fig. 3A). AOPi eventually undergoes conversion to AOPm in both normal and diabetic rats, as indicated by the 150-min chase experiment (Fig. 3). Thus it appears that the kinetics of AOPi appearance were indistinguishable from normal in the BBd rat (Figs. 2 and 3). However, there appeared to be a possible variation in the processing rate of AOP among the normal and diabetic rat groups. For instance, as indicated in Figs. 2A and 3A, conversion of AOPi to AOPm began in the Wist rat by 15 min and was almost complete by 60 min. In the BBd rat, however, AOPm was essentially undetectable by 30 min of chase (not shown), and an appreciable amount of AOPi was still relatively detected at 60 min of chase (Fig. 2B). Moreover, Fig. 3 indicates that AOPi persisted in the ERG fractions of the diabetic rat even after 150 min of chase, at which point it essentially disappeared from the ERG fractions of the normal animal. This suggests a longer half-life of AOPi in the diabetic animal. Moreover, scanning of the autoradiogram in Fig. 3 and calculating the AOPm-to-AOPi ratio as an index of maturation revealed a lower maturation level of AOP precursor in the diabetic rat compared with its normal counterpart (AOPm:AOPi in diabetic vs. normal: 0.00 vs. 0.16 at 15 min, 8.38 vs. 12.21 at 60 min, and 8.43 vs. 49.34 at 150 min of chase). Thus it appears that the conversion of AOPi to AOPm in ER-Golgi membranes is delayed in the BBd animal.
To analyze further the apparent delayed conversion of AOPi to AOPm in the BBd rat, we pulse-chased rats for 30-180 min with methionine (see MATERIALS AND METHODS). AOP was then isolated from ERG and analyzed by SDS-PAGE and autoradiography. AOPi and AOPm were excised from the dried gels (Fig. 4), and the radioactivity ratio of AOPm to AOPi was calculated (Fig. 4C). Because AOPi is converted stoichiometrically to AOPm in intact normal rats (2), we expressed the conversion of AOPi to AOPm by estimating the 50% conversion (AOPm-to-AOPi radioactivity ratio = 1:1) as an internal control to correct for any variation in label incorporation among animals. Notably, there was a lower AOPm:AOPi in BBd rats than in Wist rats at all periods of chase (Fig. 4C). The 1:1 ratio of AOPm to AOPi occurred at ~30 min of chase in the Wist rat but was not achieved until 60-90 min in the diabetic BBd rat (Fig. 4). As maturation proceeded, AOPm became the predominant species for both normal and diabetic rats, but even at 180 min of chase, the AOPm:AOPi for the diabetic animal achieved one-half the value of that of the normal animal (3.3 vs. 6.8, Fig. 4C). Statistical analysis by the t-test revealed that these differences between the animal groups were significant (P < 0.05). Also, a plot of the maturational AOPm:AOPi vs. the chase time produced linear regression curves (not shown, R2 = 0.936 for Wist rats and 0.829 for BBd rats) with significantly different slopes (0.019 for Wist rats and 0.004 for BBd rats; P < 0.01); this verified the delayed maturation of AOP in the diabetic animal. The reduced AOPm:AOPi at each time period in BBd rats is consistent with the relative persistence of AOPi, caused by a slower processing of the high mannose, a delay in exit from the ER, or both in the BBd rat.
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Abnormal jejunal AOP in type 2 diabetes.
Jejunal AOP was immunoprecipitated from detergent-solubilized BBM of
normal and diabetic female subjects and analyzed by 7% acrylamide
electrophoresis (Fig. 5). AOP from
diabetic subjects migrated as a 5-kDa smaller structure than normal
(135 vs. 140 kDa). Similar observations were made in male subjects
(data not shown).
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DISCUSSION |
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Similar to our previous studies in the congenitally diabetic BBd rat (23), we have observed in the current studies that the brush-border pool of AOP in humans with type 2 diabetes is smaller (~5 kDa) than normal. Thus it appears that abnormal AOP structure is common in diabetes.
Furthermore, chemical removal of all carbohydrate chains by trifluoromethanesulfonic acid suggested that the reduced mass of the intact AOP glycoprotein in the BBd rat appears to involve structural changes in its carbohydrate structure in diabetes (23). Because this alteration was also present in the newly synthesized AOP within hours after administration of a radiolabeled precursor, structural alterations either during intracellular synthesis and assembly or shortly after insertion into the BBM were suggested. Nonenzymatic glycosylation (21) that occurs slowly over days to weeks as a function of the elevated extracellular glucose concentrations in diabetes seemed an unlikely mechanism because of the rapid nature of the mass change (only 3 days after the onset of diabetes) and the lack of correlation with plasma glucose levels (23).
Change in AOPm mass is caused by alteration of its N-linked chains. In this report, we have examined the intracellular processing of the AOP precursor in intact rats as it is synthesized and assembled in association with intracellular membranes and then inserted into the brush-border surface membrane of the enterocyte. The apoprotein molecular mass, estimated after deglycosylation of AOPi with Endo H, was the same in BBd and Wist rats. This suggests that protein synthesis and cotranslational assembly of high-mannose N-linked chains of AOPi proceeded rapidly and normally in the BBd rat (Figs. 2 and 3). These findings might have been expected in diabetes, given the normal synthesis (27) and activity of dolichol phosphate-mannosyl and N-acetylglucosamine transferases of membrane glycoproteins reported in hepatic tissues from patients with diabetes mellitus (3).
However, conversion of AOPi to AOPm in the Golgi compartment, involving maturation of N-linked chains into complex forms and addition of O-linked chains, was delayed and resulted in a AOPm product that was smaller in BBd (135 kDa) than in Wist (140 kDa) rats (Table 1). Removal of all N-linked chains from AOPm by PNGase F suggested that this structural alteration was due to a reduction in mass of its N-linked chains during the conversion of AOPi to AOPm in the Golgi compartment, with no accompanying change of its O-linked glycans. In contrast to the Wist rat, in which the precursor essentially underwent complete maturation in the Golgi compartment, in support of previous reports (2, 11), less than one-third of the N-linked carbohydrates remained immature or included hybrid high-mannose structures in the BBd rat (Figs. 1 and 2). Despite a lack of complete maturation, AOPm was transported to its final destination in the brush-border surface membrane in a manner similar to that in the Wist rat (Figs. 1 and 3). This is consistent with detection at the cell surface of sucrase (12), insulin and insulin-like growth factor I receptors (14), and pp120, a substrate of the insulin receptor tyrosine kinase (10) in organ and cell culture systems. Our results are in agreement with those of Chapman et al. (9), who found that theDelayed maturation of N-linked chains in diabetes.
Although AOPi in the BBd rat initially appeared
rapidly (15 min) and displayed a normal mass on SDS gels, its
subsequent conversion to AOPm was prolonged, and its
half-conversion time to AOPm (1:1 labeling ratio of
AOPi:AOPm) was delayed by approximately two- to
threefold (Fig. 4C; 60-90 min in BBd vs. 30 min in Wist). The retardation of AOP maturation in the BBd
rat may involve several mechanisms. Among these are 1)
altered activity of glycosidases, mannosidases, and transferases
responsible for the modification to complex N-linked chains,
2) changes in the concentration of glycosyl substrates,
3) alternative routing by possibly bypassing the Golgi
apparatus, and 4) prolonged residence time in ER. Whether the apparent alterations in N-linked carbohydrate composition of AOP in
congenital diabetes are a consequence of changes in glycosyl substrates
or altered processing enzymes (glycosidases or glycotransferases)
necessary for the processing of AOP carbohydrates awaits detailed
structural analysis of these carbohydrate chains and of the enzymes and
substrates required for N- and O-linked glycan assembly. Whether some
AOPi bypass the Golgi compartment in diabetes on their exit
from the ER is at the moment unknown, but this is possible based on the
significant role of N-linked carbohydrates in the proper
compartmentalization and intracellular routing of glycoproteins
(8). Moreover, a default pathway for the transport of
membrane proteins from ER to Golgi has been described (25). For instance, some proteins may return to the ER
after exiting the Golgi by a yet-unknown mechanism. How much of these proteins may shuttle back to the Golgi is not clear. Prolonged residence time in ER of aquaporin-2 has been reported to occur in
nephrogenic diabetes insipidus (30). Moreover,
site-directed mutagenesis of some N-glycosylation sites in
the -chain of the insulin receptor led to accumulation of the
receptor precursor in ER (4, 6). Thus accumulation of AOP
precursor in ER might occur in congenital diabetes, perhaps among other
factors, as a result of inefficient removal of the three nonreducing
terminal glucose residues by
-glucosidase I or II, a step that may
be a prerequisite for glycoprotein transport from the ER to Golgi (26). Because the ER and Golgi fractions were of necessity
pooled together (ER-Golgi) in our experiments, we cannot distinguish between prolonged residence in the ER and a reduced rate of vesicular mediated transport of the AOPi from the ER to the Golgi
compartment. Similar to our observations, Koh et al. (18)
reported delayed processing of the insulin proreceptor to mature
insulin receptor in hepatocytes derived from STZ-diabetic rats. Thus
coordinate changes in the kinetics of intracellular assembly of
membrane glycoproteins may constitute a common phenomenon in diabetes.
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ACKNOWLEDGEMENTS |
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The authors thank Nilda A. Santiago for determination of the apparent molecular mass of the AOP species on SDS gels, Jia-Shi Zhu for helpful suggestions and assistance with figures, and Dr. Pierre Thibert and his colleagues at the Health Protection Branch, Health and Welfare, Ottawa, ON, Canada, for providing the BB rats.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Research Grants DK-11270 and DK-35033 to G. M. Gray and DK-54254 and DK-57497 to S. M. Najjar, an institutional training grant in academic gastroenterology (DK-07056), and Digestive Disease Center Grant DK-38707.
Address for reprint requests and other correspondence: S. M. Najjar, Medical College of Ohio, 3035 Arlington Ave., Block Health Science Bldg., Rm. 270, Toledo, Ohio, 43614 (E-mail: snajjar{at}mco.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 April 2000; accepted in final form 24 July 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ahnen, DJ,
Santiago NA,
Cézard J-P,
and
Gray GM.
Intestinal aminooligopeptidase. In vivo synthesis on intracellular membranes of rat jejunum.
J Biol Chem
257:
12129-12135,
1982
2.
Ahnen, DJ,
Mircheff AK,
Santiago NA,
Yoshioka C,
and
Gray GM.
Intestinal surface aminooligopeptidase. Distinct molecular forms during assembly on intracellular membranes in vivo.
J Biol Chem
258:
5960-5966,
1983
3.
Alhadeff, JA,
and
Watkins P.
Dolichyl phosphate-mannosyltransferase and dolichyl phosphate-N-acetylglucosaminyltransferase activities in liver preparations from normal controls and patients with cystic fibrosis and diabetes mellitus.
Clin Chim Acta
134:
1-9,
1983[ISI][Medline].
4.
Bastian, W,
Zhu J,
Way B,
Lockwood D,
and
Livingston J.
Glycosylation of Asn397 or Asn418 is required for normal insulin receptor biosynthesis and processing.
Diabetes
42:
966-974,
1993[Abstract].
5.
Berenson, GS,
Radhakrishnamurthy B,
Dalferes ER, Jr,
Ruiz H,
Srinivasan SR,
Plavidal F,
and
Brickman F.
Connective tissue macromolecular changes in rats with experimentally induced diabetes and hyperinsulinism.
Diabetes
21:
733-743,
1972[ISI][Medline].
6.
Caro, LH,
Ohali A,
Gorden P,
and
Collier E.
Mutational analysis of the NH2-terminal glycosylation sites of the insulin receptor -subunit.
Diabetes
43:
240-246,
1994[Abstract].
7.
Chandramouli, V,
and
Carter JR.
Cell membrane changes in chronically diabetic rats.
Diabetes
24:
257-262,
1975[Abstract].
8.
Chaney, W,
Sundaram S,
Friedman N,
and
Stanley P.
The Lec4A CHO glycosylation mutant arises from miscompartmentalization of a Golgi glycosyltransferase.
J Cell Biol
109:
2089-2096,
1989[Abstract].
9.
Chapman, AE,
Copeland P,
Davidson S,
and
Calhoun JC IV.
Diabetic BB/Wor rat haptoglobin exhibits a probable structural abnormality in Asn-linked oligosaccharides.
Biochim Biophys Acta
1077:
265-272,
1991[ISI][Medline].
10.
Choice, CV,
Poy MN,
Formisano P,
and
Najjar SM.
Comparison of the intracellular trafficking of two alternatively spliced isoforms of pp120, a substrate of the insulin receptor tyrosine kinase.
J Cell Biochem
76:
133-142,
1999[ISI][Medline].
11.
Danielsen, EM.
Biosynthesis of intestinal microvillar proteins.
Biochem J
204:
639-645,
1982[ISI][Medline].
12.
Danielsen, EM,
and
Cowell GM.
Biosynthesis of intestinal microvillar proteins. Processing of N-linked carbohydrate is not required for surface expression.
Biochem J
240:
777-782,
1986[ISI][Medline].
13.
Dennis, JW,
Granovsky M,
and
Warren CE.
Protein glycosylation in development and disease.
Bioessays
21:
412-421,
1999[ISI][Medline].
14.
Duronio, V,
Jacobs S,
and
Cuatrecasas P.
Complete glycosylation of the insulin and insulin-like growth factor I receptors is not necessary for their biosynthesis and function. Use of swainsonine as an inhibitor in IM-9 cells.
J Biol Chem
261:
970-975,
1986
15.
Gray, GM,
and
Santiago NA.
Intestinal surface aminooligopeptidases. I. Isolation of two weight isomers and their subunits from rat brush border.
J Biol Chem
252:
4922-4928,
1977[ISI][Medline].
16.
Jacobs, LR.
Alterations in labeling of cell surface glycoproteins from normal and diabetic rat intestinal microvillous membranes.
Biochim Biophys Acta
649:
155-161,
1981[ISI][Medline].
17.
Kania, RK,
Santiago NA,
and
Gray GM.
Intestinal surface amino-oligopeptidases. II. Substrate kinetics and topography of the active site.
J Biol Chem
252:
4929-4934,
1977[ISI][Medline].
18.
Koh, G,
Robinson KA,
and
Buse MG.
Delayed processing of the insulin proreceptor by hepatocytes from diabetic rats.
Biochem Biophys Res Commun
204:
725-731,
1994[ISI][Medline].
19.
Konrad, RJ,
Janowski KM,
and
Kudlow JE.
Glucose and streptozotocin stimulate p135 O-glycosylation in pancreatic islets.
Biochem Biophys Res Commun
267:
26-32,
2000[ISI][Medline].
20.
Marliss, EB,
Nakhooda AF,
and
Poussier P.
Clinical forms and natural history of the diabetic syndrome and insulin and glucagon secretion in the BB rat.
Metabolism
32:
11-17,
1983[ISI][Medline].
21.
Means, GE,
and
Chang MK.
Nonenzymatic glycosylation of proteins. Structure and functions changes.
Diabetes
31, Suppl. 3:
1-4,
1982[Abstract].
22.
Najjar, SM,
Hampp LT,
Rabkin R,
and
Gray GM.
Sucrase--dextrinase in diabetic BioBreed rat: reversible alteration of subunit structure.
Am J Physiol Gastrointest Liver Physiol
260:
G275-G283,
1991
23.
Najjar, SM,
Hampp LT,
Rabkin R,
and
Gray GM.
Altered intestinal and renal brush border amino-oligopeptidase structure in diabetes and metabolic acidosis: normal and biobreed (BB) rats.
Metabolism
41:
76-84,
1992[ISI][Medline].
24.
Nguyen, TD,
Broyart J-P,
Ngu KT,
Illescas A,
Mircheff AK,
and
Gray GM.
Laterobasal membranes from intestinal epithelial cells: isolation free of intracellular membrane contaminants.
J Membr Biol
98:
197-205,
1987[ISI][Medline].
25.
Townsley, FM,
and
Pelham HR.
The KKXX signal mediates retrieval of membrane proteins from the Golgi to the ER in yeast.
Eur J Cell Biol
64:
211-216,
1994[ISI][Medline].
26.
Pfeffer, SR,
and
Rothman JE.
Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi.
Annu Rev Biochem
56:
829-852,
1987[ISI][Medline].
27.
Sharma, C,
Dalfares ER, Jr,
Radhakrishnamurthy B,
DePaolo CJ,
and
Berenson GS.
Hepatic glycoprotein synthesis in streptozotocin diabetic rats.
Biochem Int
15:
395-401,
1987[ISI][Medline].
28.
Srinivasan, SR,
Berenson GS,
and
Radhakrishnamurthy B.
Glycoprotein changes in diabetic kidneys.
Diabetes
19:
171-175,
1970[ISI][Medline].
29.
Stieger, B,
Matter K,
Baur B,
Bucher K,
Höchli M,
and
Hauri H-P.
Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface.
J Cell Biol
106:
1853-1861,
1988[Abstract].
30.
Tamarappoo, BK,
and
Verkman AS.
Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones.
J Clin Invest
101:
2257-2267,
1998
31.
Tauc, M,
Chatelet F,
Verroust P,
Vandewalle A,
Poujeol P,
and
Ronco P.
Characterization of monoclonal antibodies specific for rabbit renal brush-border hydrolases: application to immunohistological localization.
J Histochem Cytochem
36:
523-532,
1988[Abstract].
32.
Watt, VM,
and
Yip CC.
Amino acid sequence deduced from a rat kidney cDNA suggests it encodes the Zn-peptidase aminopeptidase N.
J Biol Chem
264:
5480-5487,
1989
33.
Williamson, DH,
and
Mellanby J
D-()-3-Hydroxybutyrate.
In: Methods of Enzymatic Analysis, edited by Bergmeyer HU.. New York: Academic, 1984, vol. 4, p. 1836-1839.
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