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Cell, Vol. 46, 227-234, July 18, 1966, Copyright 0 1986 by Cell Press
The Sucrase-lsomaltase Complex: Primary Structure,Membrane-Orientation, and Evolution of a Stalked,Intrinsic Brush Border Protein
Walter Hunziker,“t Martin Spiess,
Giorgio Semenza,t and Harvey F. Lodish**
* Whitehead Institute for Biomedical Research
Nine Cambridge Center
Cambridge, Massachusetts 02142
tDepartment of Biochemistry
Swiss Federal Institute of Technology
CH-8092 Zurich
Switzerland
*Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
Summary
The complete primary structure (1827 amino acids) of
rabbit intestinal pro-sucrase-isomal tase (proSI) was
deduced from the sequence of a nearly ful l-length
cDNA. Pro-S1 is anchored in the membrane by a single
20 amino acid segment spanning the bilayer only
once. The amino-terminal, cytoplasmic domain con-
sists of 12 amino acids and is not preceded by a
cleaved leader sequence. This suggests a dual role for
the membrane-spanni ng segment as an uncleavedsignal for membrane insertion. This is foll owed by a 22
residue serine/threonine-rich, probably glycosylated,
stretch, presumably forming the stalk on which the
globular, catalytic domains are directed into the in-
testinal lumen. Following this is a high degree of
homology between the isomaltase and sucrase por-
tions (41% amino acid identity), indicating that pro-S1
evolved by partial gene duplication.
Introduction
The sucrase-isomalt ase complex (SI, EC 3.2.1.48-10) is an
intrinsic gl ycoprotein of the small intestinal brush bordermembrane and plays a key role in the final degradation of
glycogen and starch. SI is synthesized as a single chain
precursor (proSI) of approximately 260,000 daltons with
both enzymatically active sites of the mature protein (Se-
menza, 1978,1979; Hauri et al., 1979, 198O; Sjostrijm et al.,
1980; Montgomery et al., 1981; Wacker et al., 1981; re-
viewed by Semenza, 1986). Once it reaches the apical
cell surface, pro-S1 is split by pancreatic proteases into
two subunits, i somaltase (approxi mately 140,000 daltons,
originally the amino-terminal portion of proSI) and su-
erase (approximately 120,000 daltons), which remain non-
covalently associated. The complex is anchored in the
membrane by a single hydrophobi c segment located at
the amino terminus of isomaltase @runner et al., 1979;
Spiess et al., 1982). By digestion with papain more than
90% of the mass of SI is released from the membrane as
an enzymatically fully active soluble protein; only the
amino-terminal part of isomaltase remains anchored in
the bilayer.
Overlapping substrate specificity of the two subunits
(both can hydrolyze maltose and a number of other a-glu-
copyranosides) and a number of other functional and
structural similarities suggested that SI has evolved by
gene duplication (Semenza, 1978).
To study in more detail the membrane anchoring, bio-
synthesis, and phylogenetic origin of SI, we have cloned
and sequenced its cDNA. The deduced amino acid se-
quence reveals a strong homology between t he two sub-
units. Furthermore, pro-S1 has no cleaved signal and
spans the bilayer only once in an N(in)/C(out) direction,
partially correcti ng previous conclusions (Frank et al.,1978; Btirgi et al., 1982).
Results and Discussion
Nucleotide and Deduced Amino Acid Sequence
of Pro-S1
Initially, cDNA clones encoding SI were isolated by anti-
body screening of a hgtll cDNA l ibrary prepared from
rabbit small intestinal mucosa mRNA. Longer and nearly
full-length cDNAs were then obtained by hybridization
screening. Overlapping cDNA fragments of three of these
clones covering nearly 6 kb of the pro-S1 mRNA were se-
quenced by the Sangerldideoxy procedure using the shot-gun sonication strategy (see Experimental Procedures).
The combined nucleotide sequence included the com-
plete coding and 3’ noncoding region, as well as part of
the 5’ noncoding region (Figure 1).
The sequence encodes a polypeptide of 1827 residues
(Figure 2) with a calculated molecular weight of 203,000
daltons. This is in reasonable agreement with the deter-
mined apparent molecular weight of pro-S1 of approxi-
mately 260,000 daltons (Sjbstriim et al., 1980), which
includes 15% carbohydrat e (Cogoli et al., 1972). The
deduced amino acid composition correlates well with that
determined for mature SI (Cogoli et al., 1972).
The derived amino acid sequence is confirmed by itsessential agreement with that of the small fragments of SI
that have been sequenced. Besides its identity with the
amino terminus of isomaltase (Sjostrom et al., 1982), there
are only minor diff erences from the protein sequences de-
termined for the amino terminus of sucrase (starting at po-
sition 1008; Sjiistriim et al., 1982) and for the active sites
of the two subunits (around Asp-505 and 1249; Quaroni et
al., 1976).
In vivo pro-S1 is posttranslationall y cleaved by pan-
creatic prot eases (el astase in particular) to an isomaltase
and a sucrase subunit (Hauri et al., 1979,1980, 1981; Sjos-
trijm et al., 1980; Wacker et al., 1981). The experimentall y
determined sequence of the amino terminus of sucrase
(Sjostriim et al., 1982) is found (with minor discrepancies)
in the cDNA-deri ved sequence beginning with lle-1008.
Since we do not know whether only one proteolytic cut
converts pro-S1 to isomaltase and sucrase, and since
there are only scanty data on the compositi on of the car-
boxy terminus of mature isomaltase (and sucrase) (Brun-
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Structure of Pro-Sucrase-lsomaltase229
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Figure 2. Amino Acid Sequence and internal Homol ogy of Pro-S1
The amino acid sequence deduced from the cDNA sequence of Figure 1 is shown. The homologous regions of sucrase and isomaltase were aligned
as described in Experimental Procedures. Identical residues are boxed. The membrane-spanning region is underlined. The SedThr-rich, probably
0-glycosylated, domain is marked wi th a broken line. Asp-505 and Asp-1349, located in the active sites of the respective subunits, are boxed withthick lines. A vertical arrow indicates the N-terminal lie-1008 of the sucrase subunit in mature SI. I: isomaltase. S: sucrase.
ner et al., 1979) we cannot l ocate precisely the positi on
of the carboxy terminus of mature isomaltase, nor can we
rule out posttranslational processing at the C-terminus of
mature sucrase.
Carbohydrat e analysis of the mature enzyme complex
indicates both N-and O-linked glycosylati on (Cogoli et al.,
1972). The deduced amino acid sequence contains 19
potential N-glycosylation sites, Asn-X-Ser/Thr, 12 of which
are located in the sucrase portion of proSI. It is not known
how many of the potential sites are actually glycosylated.
Several Ser/Thr-rich stretches may contain O-linked sug-
ars. Starting at position 44, 17 out of 22 residues are either
Ser or Thr. Shorter Ser/Thr clusters begin at positions 120,
805, 984, and 1750.
detected a single tubulin mRNA of approximately 1800 bp
in all samples (data not shown), indicating that in those
RNA preparations at least smaller molecules were not sig-
nificantly degraded. Nevertheless, it seems likely that the
RNAs hybridi zing with pro-S1 cDNA in the lower molecular
weight region of the intestinal sample are due to partial
degradation of the large 6 kb pro-S1 mRNA. However, we
cannot rule out the existence of other RNAs homologous
to SI.
Domain Structure and Orientation of Pro-S1
in the Membrane
There are 26 cysteine residues, some of which may
form disulfide bonds within the subunits. However, su-
erase and isomaltase are not linked by disulfide bonds
@runner et al., 1979). While native SI does not possess
free thiol groups, approximately five to six are exposed
upon unfolding (Cogoli et al., 1972).
Pro-S1 cDNA Hybridizes to a 6 kb mRNA Specific
for Intestinal Mucosa
The mature SI complex is anchored in the membrane
solely by a small segment at the amino terminus of the
isomaltase subunit (Brunner et al., 1979; Spiess et al.,
1982). The sucrase subunit does not interact with the
hydrophobi c core of the membrane: it is not labeled by the
highly hydrophobic photolabel 3-trifluoromethyl-3-(m-iodo-
phenyl)diaziri ne (TID) and it is preferentially solubilized by
citraconylation of brush border membranes. Furthermore,
anti-sucrase antibody conjugates are 4-5 nm more re-
mote from the brush border membrane than those formed
by anti-isomaltase antibodies (Nishi and Takesue, 1976).
The pro-S1 cDNA hybridizes to a polyadenylated RNA Figure 4 shows a hydropathy plot (Kyte and Doolittle,
from intestinal mucosa of approximately 6 kb (Figure 3). 1982) of the cDNA-derived pro-S1 sequence. Regions of 21
No homologous mRNA were detected in rabbit mammary amino acids wi th a hydropathy average >1.5 are likely to
glands or HeLa cells, both known not to express SI. be associated with the lipid bilayer and in several proteinsReprobing of the same Northern blots with tubulin cDNA have been shown to span the membrane in a helical con-
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Cdl230
kb ABC
Figure 3. Northern Blot Analysis
Ten micrograms of poly(A) selected RNA was fractionated on a 1.5%
agarose formaldehyde denaturing gel, transferred to nitrocellulose,
and hybridized with nick-translated pro-S1 cDNA. Lane A: rabbit small
intestinal mucosa RNA. Lane 6: rabbit mammary gland RNA. Lane C:
HeLa cell RNA. End-labeled fragments of Hindlll cut EDNA were used
as size markers.
formation (Kyte and Doolittle, 1982; Eisenberg, 1984). The
absence of a sufficiently hydrophobic segment in the su-
erase porti on of pro-S1 is in agreement with the peripheral
positioning of sucrase and further indicates that there are
no additional hydrophobic anchor sequences in pro-S1
near the carboxy terminus of sucrase which might have
been severed off by proteolytic processing.
The only potential hydrophobi c anchor segment is the
sequence between residues 13 and 32 at the amino termi-
nus of isomaltase (Figures 2 and 4). This segment is
known to be embedded in the brush border membrane,
since it is labeled by the hydrophobic photoreagen t TID
(Spiess et al., 1982). Analysis of its secondary structure
by the method of Chou and Fasman (1978) predicts a heli-
cal conformation for the membrane anchor (even more so,
since it is in an apolar envi ronment; Green and Flanagan,
1978). Indeed, direct circular dichroism analysis of the ap-
proximately 40 amino acid terminal fragment of isomal-
tase left in the membrane after papain solubili zation of SI
indicates a helix content of >80% (Spiess et al., 1982). In
a helical conformation this 20 amino acid segment can
cross the membrane just once.
The 12 amino-terminal residues (containing one nega-
tive and three positive charges) p receding this membra-
nous sequence and a segment of approximately 180
amino acids just following it have an extremely low hy-
dropathy average (Figure 4) and are not expected to in-
teract with the hydrophobic core of the membrane. In addi-
tion, there is a conspicuous Ser/Thr-ri ch segment which
in all likelihood is 0-glycosylated. This region and the fol-
lowing enzymatic domains of isomaltase and sucrase are
0
,
400 800 1200 IMX) zcoo
Residue Number
Figure 4. Hydropathy Plot of the Pro-S1 Protein Sequence
Hydropathy values (Kyte and Doolittle, 1982) for a window of 21
residues were averaged and asigned to the middle amino acid of the
window. The averages were plotted wi th respect t o the position along
the protein sequence. The plot for the isomaltase portion (excluding
the amino-terminal 80 values) was superimposed to the plot for the su-
erase portion. A small gap was necessary to optimize the alignment.
undoubtedly located on the extracytoplasmic (luminal)
side of the membrane.
These observations strongly suggest a membrane to-
pology for pro-S1 in which the polypeptide chain spans the
membrane only once in an N(in)/C(out ) orientation (Figure
5). This corrects earlier models (Frank et al., 1978; Biirgi
et al., 1982; Sjijstrijm et al., 1982). Recent labeling experi-
ments by Brunner (personal communication) using intact
intestine instead of membrane vesicles (which are proba-
bly leaky; Gains and Hauser, 1984) confirm the cytoplas-
mic location of the amino terminus suggested here. The
earlier interpretati on of carbohydrat es attached to Thr-12
(Frank et al., 1978) was also in error: the amino terminus
of isomaltase that was sequenced was contaminated by
glycopeptides (possibly t runcated in or near the SerlThr-
rich segment; data not shown).
The polypeptide segment connecting the membranous
anchor with the globular catalytic domai ns contains a se-
quence extremely rich in serine and threonine residues.
The 22 residues between positions 44 and 85 consist of
nine serines and eight threonines, interrupted only by five
uncharged amino acids. Similar Ser/Thr-rich regions not
far from the transmembrane sequence have been ob-
served in glycophorin (Tomita and Marchesi, 1975; Tomita
et al., 1978) and the low-density lipoprotein receptor (Rus-
sel et al., 1984; Yamamoto et al., 1984). In both proteins
they are 0-glycosylated. That this is also the case for the
Ser.Thr-rich domain of pro-S1 is suggested by the compar-
ison of the SDS gel mobility of an amino-terminal fragment
of mature i somaltase (residues 2-95) and that of a corre-
sponding peptide translated in vitro: the former appears
as a diffuse band of higher apparent molecular weight
than the in vitro product (data not shown). We propose that
this Ser/Thr-ri ch domain forms a connecting piece (stalk)
between the membrane anchor and the bulk of the protein
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Cell232
changed one of these active si tes from an isomaltase-
maltase i nto a sucrase-maltase. Thus a long polypeptidechain was formed, carrying two similar, but not identical,
active sites. Posttranslational modification of this single,
long polypeptide chain by pancreatic proteases l eads to
the two subunits that make up the final SI complex. The
subunits remain associated with one another via interac-
tions formed during the folding of the original single chain
pro-SI. Proteolytic cleavage of pro-S1 occurs around resi-
due 1 e-1008, which is inside the sucrase portion as deter-
mined by the sequence homology (Figure 2). The cleav-
age site is in a three amino acid segment that has no
homology in the isomaltase portion. Thus, mature “iso-
maltase” contai ns part of the sucrase domain at its
C-terminus.Sucrase seems to be confined to terrestrial animals, be
they mammals, birds, or reptiles. Recently, an avian SI
was studied and no substantial difference from the mam-
malian enzymes was detected, additionally, it is also syn-
thesized as a pro-S1 and is anchored to the membrane
via the isomaltase portion (Hu, Spiess, Brunner , and
Semenza, unpubli shed data). This suggests that the se-
quence of events leading to pro-S1 from the ancestral
isomaltase took place prior to the separation of mammals
and reptiles, e.g., more than 300 million years ago. In-
terestingly, some mammalian species belonging to the
Pinnipedia, like the sea lion (Zelophus californianus),
have a long chain isomaltase with two identical catalyticsites, both splitting isomaltose and maltose, but not su-
crose (Wacker et al., 1984). This double isomaltase thus
mimmiks the ancestral “double isomaltase” but is likely to
have originated by back mutation.
Intestinal and renal glycosidases are thought to be a
group of enzymes closely related to one another. In addi-
tion to functional similarities to SI, the glucoamylase com-
plex and the P-glycosidase (lactase-gl ucosylceramidase)
complex have two active sites each and are synthesized
as a single chain precursor and posttranslationall y split
into the two constituent subunits of the mature forms
(Danielsen et al., 1981, 1984; Skovbjerg et al., 1982, 1984;
Siirensen et al., 1982). The glucoamylase complex isprobably anchored by the amino terminus of the pro-form
(Nor& et al., 1986). It is therefore likely that all of these
glycosidases share structural similarities with SI. It would
not be surprising, for instance, if a Ser/lhr-rich sequence
carrying O-linked carbohydrat es turns out to be a struc-
tural unit common to all the stalks of these brush border
glycosidases.
A number of proteases and peptidases of the renal and
intestinal brush border membrane have also been sug-
gested to be anchored to the lipid bi layer not far from the
amino terminus of the polypeptide chain (e.g., dipeptidyl-
peptidase IV; MacNair and Kenny, 1979; Booth and Kenny,
1980; aminopeptidases N; Booth and Kenny, 1980; Nor&
and Sjbstrbm, 1980; Ferracci et al., 1982; aminopepti dase
A; Booth and Kenny, 1980; y-glutamyl-transpeptidase;
Frielle and Curthoys, 1983; Matsuda et al., 1983) and may
well be positioned in a way similar to that we propose for
SI and may also lack a cleaved signal sequence for mem-
brane insertion. In fact, this has been shown very recentl y
for y-glutamyl-transpeptidase (Laperche et al., 1986). For
a recent revi ew see Semenza (1986).
Experimental Procedures
MaterialsGoat anti-rabbitSI serum and small intestinalmucosa poly(A)+RNAfrom a single rabbit (strainNew ZealandWhite)were a kind gift fromDr. H. Wackerand i? Huber . Reverse ranscriptasewas purchasedfrom BoehringerMannheim;all other enzymeswere from BethesdaResearchLaboratories,New EnglandEiolabs,or PharmaciaLabora-tories.
Sequencing eagentswere obtained rom PharmaciaLaboratories,deoxycytidine5’-(a-32P)-triphosphate,eoxyadenosine ’-(a-%-thio-triphosphate nd L-[4-5-3H]-leucinerom Amersham,biotinylated ab-bit anti-goat gGantibodiesand avidin-conj ugated orseradish eroxi-
dase (Vectastain) from Vector Laboratories.
General Methodsisolation of phage and plasmid DNA, restriction enzyme digestions,
agarosegel electrophoresis,Southern blot analysis and radioactivelabeling of DNA were carried out by standard procedures (Maniatis et
al., 1982). Poly(A)+ RNA for Northern blots was fractionated on 1.5%
agarose formaldehyde denaturing gels and transferred to nitrocellu-
lose (Maniatis et al., 1982). RNA blot hybridization was performed as
described by Zuker and Lodish (1981).
Intestinal Mucosa cDNA Library
We prepared a cDNA li brary from rabbit small intestinal mucosa mRNA
in the expression vector hgtll (Young and Davis, 1983). cDNA was syn-
thesized essentially as described by Gubler and Hoffman (1983). Ten
micrograms of poly(A)+ RNA primed with oligo(dT) was copied with re-
verse transcriptase. RNAase H and DNA polymerase I were used to
synthesize the second strand, omitti ng E. coli ligase. After methylati onof endogenous EcoRl sites, we blunt-end ligated the cDNA to EcoRl
linkers. Excess li nkers were digested and separated from the cDNA by
gel filtration on Sephadex G-100. The cDNA was size-fractionated by
agarose gel electrophoresis and the fraction >2.5 kb was eledroeluted
to enrich for clones encoding pro-S1 sequences and ligated into the
EcoRl site of the expression vector Xgstll (Young and Davis, 1983).
Screening of the cDNA Library
The recombinant Xgstll DNA was packaged in vitro into phage particles
(Maniatis et al., 1982). Recombinant clones expressing a b-galac-
tosidase-SI fusion protei n were detected using a 1:2500 dilution of goat
anti-rabbit SI serum (preadsorbed on an immobili zed E. coli lysate),
biotinylated rabbit anti-goat I gG antibodies, and avidin-conjugated
horseradish peroxidase (Vectastain) exactly as described by Young et
al. (1985). In situ plaque hybridizati on was carried out as described by
Maniatis et al. (1982). Positive plaques were purified to homogeneityand their cDNA insert was isolated and subcloned into the plasmid
vector pUC13 (Vieira and Messing, 1982a).
Screening of approximately 20,000 recombinant phages with antise-
rum raisedagainst SI lead to the identificationof 11 mmunoreactiveclones. Two of these clones, I.Sl-42 and ASI-46, were purified and ana-
lyzed further. Upon digestion with EcoRI, they yielded a cross-hybrid-
izing insert of approximately 1.9 kb. In addition, XSl-42 harbored asec-
ond fragment of 1.5 kb and 1SI-46 one of 3 kb that hybridi zed to each
other, suggesting that Ihe cloned cDNA possesses an internal EcoRl
site.
The nucleotide sequence of the two EcoRl fragments of LSI-42 con-
firmed the identity of the clones since they contain sequences corre-
sponding to the amino acid sequences of part of the active site
(Quaroni and Semenza, 1978) and of the amino t erminus of sucrase
(Sjbstrbm 81 al., 1982).
We used the 5’ 650 bp Hindlll fragment of the hSI-46 insert to
rescreen the cDNA library by in situ plaque hybridization and isolated
several full-length clones (X1-111, ASI-115, 151-128. and 1%138). The
missing 5’ sequence was derived from the 2 kb BamHl fragment of
clone 1%115.
The inserts of the different clones sequenced derived in all likeli-
hood from a common transcript since all of the overlapping regions se-
quenced were identical.
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Structure of Pro-Sucrase-lsomaltase233
Sequence Analysis
DNA sequencing was done by a modified procedure of the Sanger
dideoxy chain terminati on method (Sanger et a/., 1977. Random DNA
fragments created bysonication of self-ligated inserts (Deninger, 1963)
were subcloned into the Smal site of M13mp6 (Viei ra and Messing,
1962b) and sequenced according to Biggin eta/. (1983). The sequence
was assembled using the database programs of Staden (1962). The
database contained 169 gel readings representing an average nucleo-
tide redundancy of approximately five, both strands being covered
throughout the entire sequence. A Sstl/Sall f ragment containing the in-
ternal EcoRl site was sequenced starting at the Sstl site to confirm that
EcoRl doe s not cut out an additional fragment.
The alignment of homologous sequences (Figure 2) was optimized
using t he Align progr am by Dayhoff et al. (1963) with the mutational
data matrix scoring system. A break was assigned a penalty of 12.
The computing was done using the facility of Whitaker College
(MIT).
We thank Dr. H. Wacker and P. Huber for their kind gift of anti-sucrase-
isomaltase serum and small intestinal pol y(A)’ RNA and Dr. K.
Mostov for rabbit mammary gland RNA. We also thank Drs. J. Brunner
and H. Wacker for helpful discussions. We acknowledge the help of M.
Boucher with the manuscript. This work was supported by grants from
the Swiss National Science Foundation and the National Institutes of
Health. M. S. has been supported by fellowships from the Swiss Na-
tional Science Foundation and the European Molecular Biology Orga-
nization.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 16 U.S.C. Section 17 34
solely to indicate this fact.
Received March 24, 1966; revised May 1, 1966
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