86 Hunziker Cell

8/8/2019 86 Hunziker Cell

http://slidepdf.com/reader/full/86-hunziker-cell 1/8

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-



*

-2

AA

A

GC

A

1 AGC

AGA

GC

A

A

GAGrC

GrGAA~

C

GCCAGCG~Gr~C

AA

Cr~GAA

GT

T

AA~

A

CGrCrACG~

rGACA

GAG~GrCGAAAA

GC

A

C

GCAGrGC

A

GCGrGGAA

GGA

CAC

GGrGCC~GrAA

CGGCA

Gr~GAGAGA

A

T

GA

AC

GT

GA

A

~GGAGAAA

GrCCC

GA

rCA

C

CrAGrC

AC

A

AAGArCCC

GrGA

AGCGGAGCCAGAA

AAGAGrA

GrA

A

C

AC

A

T

GA

T

GAGrGA

CCGGrC

GGrGrA

GA

~

A

ACC

AC

rGArAGrAGGr

T

GAA

T

AACrCGAAAGGA

GGCACACGGAC

GAGA

AAAGGCCA

T

TT

AA

AGGA

rCGGrGr

AGA

CGCGGA

CC

CAC

ArA

AAArACGrGGCC

C

A

GAA

A

GrGGrC

rAC

ACCGGAA

CGC

CAGGArCGGAC

GArCCGG

A

ATT

AGrGGrAAA

GGACCCA

GAA

rCrGAAGAAAGGAA

A

AC

A

GA

ATGC

GGC

GAGrC

AGCGA

GGA

AAGrAACGGA

GC

CAAAGCrGC

rGGAAC

T

A

A

AGA

C

GrGrGGGrAGArCAGGAA

ArGGAAGrCGGC

GAA

rAACGAC

rC

A

C

A

GTGGC

A

C

ArGAAGAGGAGGAGA

GAGA~GrC

CGrC

GC

GGCGr

A

A

A

A

CGAAGrGAACCGrAC

GCGGACGrGCrAGGGGA

rAGAGrC

C

C

GA

C

GCAAC

GA

ACGrAA

ArC

A

AACCCA

GrC

GCGGArGGAAGCGC

TGC

GAAA

GC

GGGA

GGArGGrCACGGGAGCAArCGrGrGGAGCGGrGGrGCAACGrGGACGGCGA

A

AAC

CAGTGAC

GGGGC~~CrC

ACAGCGAGGA~GACCGAC

C

GGA

AC

TGT

T

GC

ACCAA

AGC

CCAACA~

C

GCrGGAA

rAC

GC

rCCGA

T

AGA

CGGGrGAGA

AAr~AGGGGC

CACACGrGA

GGACAA

rGArGCA

CGAGC

GT

GT

A

A

GTC

GCGGAA

C

GGrGGAGArCC

CA

AGAGCCAAGAGrA

AC

C

C

CT

C

TT

A

AGAAAGCAGAGAA

CrAGAA

CGGGAGAGGAA

A

AA

GT

A

T

CT

A

AAC

GC

GAAACAGA

ACCAAA

C

GGGTAGAA

T

TGCA

ACrCGArACCA~

AGAC

C

GrCCAGAACC

T

GAA

AT

GGA

GGAC

CAA

CA

AAGrAC

AGCA

GC

CGA

rGCC

rGGAGr

A

GGA

A

TA

AC

AC

GAGrACA

GA

ACrACC

CrAC

AGGrAA

CGAC

TA

A

A

A

CGA

A

CCrGrGGAGrGAAC

GAAGGrA

rAA~GAC

AA

AT

GT

A

C

C

GA

A

AGAGrGAC

GA

GG~

ACAGAAC

A

GA

T

GGA

T

GT

CAGACrCC

ACACCGCCGAAAAAGGrGGAACA

GC

AAA

A

GC

GGGGGGAGrC

AA

GGrA

ACAGGACCAAGGCCGGAAGAGA

AGC

GAT

T

A

C

CGCGGAGrA

CGC

C

CCA

ACrGrAGGAGACGGA

AAGrCAGC

AC

ArTC

AT

GCC

rCGC

rAGGrCAGAC

AGrCGrAGGAA

AACAAA

GA

AGGGAGTGCCA

AGrCA

AAGAAGGA

GC

AAA

GAGA

CrGA

CC

TGTA

A

AGAAGA

AA

ACGGA

C

CGAGA

GCA

GCGA

AGGGACA

AGrCC

GrC

GC

TA

TGGC

GGC

GrGGC

AGCAAA

AAGA

rCAGAAGACGrA

CC

ACC

C

GCTC

A

GA

CArGGrGGA

AA

AACA

AAAGArGAGGCGrGGAGAAGAGA

C

r

rGTA

GA

T

CA

AA

ACA

AC

AC

A

A

GA

AGGAGC

CA

AGrGCGGA

GACA

TAGA

T

AGrC

CCAGGAGGrC

C

CAAGAGCGCAA

GGC

AGGAA

GrAT

T

CGGCAGGCAGGGCGA

GGCGGAA

AGC

GrGGGA

AGGA

CACGGrAGAGGAArCC

rGGA

GACA

TGT

CGACAACCCGrACCGGA

~GGAC

AC

GC

AC

C

A

AA

A

T

GA

GrGAGA

GAGrCGAAAAAA~A

ACAA

GCGA

CGCC

GTGC

T

A

C

ArCGAGA

A

GGGAACA

rCGrGGGGrC

CAGGrA

GrGrG

GA

T

TT

TGC

T

CCGrGGrGAACAGGAAAAGGACAG4

rC

AA

CAGAGC

AAC

T

TGTGT

T

GA

GCCA

~ArCA

ArAGACCGrGCGCAGAAC

GGC

C

GACCCCGGAAGGGA

A

AGAAAAGrAACrA

AAAA

GA

C

CAGA

GGr

rAA

A

A

GA

T

rAGGGGCAGA

GACGArAAGrAAGA

C

AAA

A

C

AA

A

AGC

GArACAAGA

AACCGGrC

TAA

C

AT

CT

GA

AAAAATTAC

AGrC

GCCAArCAA

AAGrA

ACAGrGA

rAGCCArAGCA

AArTA

TC

GCArAGAAAAAGrC

AGrACGCA

rGrGAArAGAAGrAA

GrGrGA

GA

CA

rGA

GrAGrAGrA

AAA

CA

ArAAArAGrA

CGr

GrC

AGA

GT

T

AA

A

AGrAA

AA

5

Fge1NedS

oPoS

Nmbsohgncenedpo

(sapnshnaoc

Tpy

ao

sg

isu

n

Tsoc

lmintho

renfameaencebaesS

-1 1 2 3 4 6 7 8 9 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5



Structure of Pro-Sucrase-lsomaltase229

1,

I:s.

I.S:

I:s:

I:S:

I:s:

I:s:

I:s

I:s

8:

I:S:

1.s.

Ez

Is

s:

I:s:

I:s:

CRNFRVQW

LDDPIEISWS

60

117986

1741046

2331106

2931164

3531224

4131283

4701340

5111400

5631460

6231519

6831577

7431637

8031697

8631757

9231817

9311827

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-



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



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.



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

References

Biggin, M. D., Gibson, T. J., and Hong, G. F. (1963). Buffer gradient gels

and % label as an aid to rapid DNA sequence determination. Proc.

Natl. Acad. Sci. USA 60, 3963-3965.

Booth, A. G., and Kenny, A. J. (1960). Proteins of the kidney microvillar

membrane. Asymetric labeling of the membrane by lactoperoxidase-

catalyzed radioiodination and by photolysis of 35di(tz51)iodo-4-

azidobenzenesulphonate. Biochem. J. 167, 31-44.

Bos, T J., Davis, A. R., and Nayak, D. F! (1964). NHs-termi nal

hydrophobic region of influenza virus neuraminidase provides the sig-

nal function in translocation. Proc. Natl. Acad. Sci. USA67,2327-2331.

Braun. H., Legler, G., Dechusses, J., and Semenza, G. (1977).

Stereospecific ring opening of conduritol-B-epoxide by an active site

aspartate residue of sucrase-isomaltase. Biochym. Biophys. Acta 483,

135-l 40.

Brunner, J., Hauser, H., Braun, H., Wilson, K. J., Wacker, H., O’Neill,

B., and Semenza, G. (1979). The mode of association of the enzyme

complex sucrase-isomaltase with the intestinal brush-border mem-

brane. J. Biol. Chem. 254, 1621-1626.

Burgi, R., Brunner, J., and Semenza, G. (1983). A chemical procedure

for determining the sidedness of the NH2 terminus in a membrane

protein. The small intestinal sucrase-isomaltase. J. Biol. Chem. 258,

15114-15119.

Chou, P Y., and Fasman, G. D. (1976). Empirical predictions of protein

conformation. Ann. Rev. Biochem. 47; 251-276.

Cogoli, A., and Semenza, G. (1975). A probable oxocarbonium ion in

the reaction mechani sm of small intestinal sucrase and isomaltase.J. Biol. Chem. 250, 7602-7609.

Cogoli, A., Mosimann, H., Vock, C., von Balthazar, A.-K., and

Semenza, G. (1972). A simplified procedure for the isolation of the

sucrase-isomaltase complex from rabbit intestine. Its amino-acid and

sugar composition. Eur. J. Biochem. 30, 7-14.

Cowell, G. M., Sjostrom, H., Noren, O., and Tranum-Jensen, J. (1966).

Topology and quarternary structure of pro-sucrase-isomaltase and fi-

nal form sucrase-isomaltase. Biochem. J., in press.

Danielsen, E. M., Skovbjerg, H., Noren, O., and Sjiistrom, H. (1961).

Biosynthesis of microvillar proteins. Nature of precursor forms of

microvillar enzymes from Gas+-precipitated enterocyte membranes.

FEES Lett. 732, 197-200.

Danielsen, E. M., Skovbjerg, H., Noren, O., and Sjdstriim, H. (1964).

Biosynthesis of intestinal microvillar proteins. Intracellular processing

of lactase-phlorizin hydrolase. Biochem. Biophys. Res. Commun. 722.

62-90.

Dayhoff, M. O., Barker, W. C., and Hunt, T. L. (1963). Establishing ho-

mologies in protein sequences. Meth. Enzymol. 97, 534-545.

Deninger, P L. (1963). Random subcloning of sonicated DNA: applica-

tion to shotgun DNA sequence analysis. Anal . Biochem. 129,216-223.

Eisenberg, D. (1964). Three-dimensional structure of membrane and

surface proteins. Ann. Rev. Biochem. 53, 595-623.

Engelman, D. E., and Steitz, T. A. (1961). The spontaneous insertionof proteins i nto and across membranes: the helical hairpin hypothesis.

Cell 23, 411-422.

Feracci, H.. Maroux, S., Bonicel. J., and Desnuelle, P (1962). The

amino acid sequence of the hydrophobic anchor of rabbit intes-

tinal brush border aminopeptidase N. Biochem. Biophys. Acta 684,

133-136.

Fields, S., Winter, G., and Brownlee, G. C. (1961). Structure of the neu-

raminidase gene in human influenza virus A/PR/6/34. Nature 290.

213-216.

Frank, G., Brunner, J., Hauser, H., Wacker, H., Semenza, G., and

Zuber, H. (1976). The hydrophobic anchor of small-intestinal sucrase-

isomaltase. N-terminal sequence of the isomaltase subunit. F EBS

Lett. 95, 163-166.

Frielle, T., and Curthoys, N. I? (1963). Characterization of the mem-

brane binding domain of gamma-glutamyltranspepti dase by specificlabeling techniques. Biochemistry 22, 5709-5714.

Gains, N., and Hauser, H. (1964). Leakiness of brush border vesicles.

Biochem. Biophys. Acta 772, 161-166.

Green, M. N., and Flanagan, M. T. (1976). The prediction of the confor-

mation of membrane proteins from the sequence of amino acids. Bio-

them. J. 153, 729-732.

Gubler, U., and Hoffman, B. J. (1963). A simple and very efficient

method f or generating cDNA li braries. Gene 25, 263-269.

Halegoua, S., and Inouye, M. (1979). Secretion of outer membrane pro-

teins from E. coliacross the cytoplasmic membrane. In Bacteria/ Outer

Membranes, M. Inouye, ed. (New York: John Wiley&Sons), pp. 67-114.

Hauri, H. P, Quaroni, A., and Isselbacher, K. J. (1979). Biogenesis of

intestinal plasma membrane: posttranslational route and cleavage of

sucrase-isomaltase. Proc. Natl. Acad. Sci. USA 76, 5163-5166.

Hauri, H. P, Quaroni, A., and Isselbacher, K. J. (1960). Monoclonal an-

tibodies t o sucrase-isomaltase: probes for the study of postnatal devel-

opment and biogenesisof the microvillus membrane. Proc. Natl. Acad.

Sci. USA 77, 6629-6633.

Hauri, H. P, Wacker, H., Rickli, E. E., Bigler-Meier, B., Quaroni, A., and

Semenza, G. (1962). Biosynthesis of sucrase-isomaltase. Purification

and NHp-terminal amino acid sequence of the rat sucrase-isomaltase

precursor (pro-sucrase-isomaltase) from fetal intestine transplants.

J. Biol. Chem. 257, 4522-4526.

Holland, E. C., Leung, J. O., and Drickamer, K. (1964). Rat liver

asialoglycoprotein receptor lacks a cleavable NH*-terminal signal se-

quence. Proc. Natl. Acad. Sci. USA 87, 7336-7342.

Kyte, J., and Doolittle, R. F. (1962). A simple method for displaying the

hydrophobic character of a protein. J. Mol. Biol. 157, 105-132.

Laperche. Y., Bulle, F., Aissani, T., Chobert. M.-N., Aggerbeck, M.,

Hanoune, J., and Guellaen, G. (1986). Molecular cloning and nucleo-tide sequence of the rat kidney y-glutamyl transpeptidase cDNA. Proc.

Natl. Acad. Sci. USA 83, 937-941.

MacNair, R. D., and Kenny, A. J. (1979). Proteins of the kidney microvil-

lar membrane. The amphiphatic form of dipeptidyl peptidase IV. Bio-

them. J. 779, 379-395.

Maniatis, T, Fritsch, E. F., and Sambrook, J. (1962). Molecular Cloning:



Cell234

A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Har-

bor Laboratory), pp. 1-545.

Matsuda, Y., Tsuji, A., and Katunuma, N. (1983). Studies on the struc-

ture of gamma-glutamyltranspepti dase III. Evidence that the amino ter-

minus of the heavy subunit is the membrane binding segment, J. Bio-

them. Tokyo 93, 1427-1433.

Montgomery, R. K., Sybicki, A. A., Forcier, A. G., and Grand, R. J.

(1981). Rat intestinal microvill us membrane sucrase-isomaltase is a

single hi gh molecular weight protei n and fully active enzyme in the ab-

sence of luminal factors. Bi ochem. Biophys. Act a 667, 346-349.

Nishi, Y., and Takesue, Y. (1976). Localization of intestinal sucrase-

isomaltase complex on the microvillus membrane by electron micros-

copy using nonlabeled antibodies. J. Cell Biol. 79, 516-525.

Noren, O., and Sjostriim, H. (1980). The insertion of pig microvillus

aminopeptidase into the membrane as probed by ‘zel-iodonaphthyl-

azide. Eur. J. Biochem. 704, 25-31.

Noren, O., Sjostriim, H., Danielsen, E. M., Cowell, G., and Skovbjerg,

H. (1986). The enzymes of the enterocyte plasma membrane, In

Molecular and Cellular Basis of Digestion, f? Desnuelle, H. Sj&trom,

and 0. Noren, eds. (Amsterdam: Elsevier), pp. 327-368.

Proudfoot, N. J., and Brownlee, G. G. (1976). S’noncoding region se-

quences in eukaryotic messenger RNA. Nature 263, 211-214.

Quaroni. A., and Semenza. G. (1976). Partial amino acid sequences

around the essential carboxylate in the active sites of the intestinal

sucrase-isomaltase complex. J. Biol. Chem. 257, 3250-3253.

Russell, D. W., Schneider, W. J., Yamamoto, T, Luskey, K. L., Brown,

M. S., and Goldstein, J. L. (1984). Domain map of the LDL receptor:

sequence homology with the epidermal growth factor precursor. Cell

37, 577-585.

Sanger, F, Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with

chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,5463-5467.

Schneider, C., Owen, M. J., Banville, D., and Williams, J. G. (1984). Pri-

mary structure of human transferrin receptor dedu ced from the mRNA

sequence. Nature 311, 675-678.

Semenza, G. (1978). The sucrase-isomaltase complex, a large dimeric

amphiphatic protein from the small intestinal brush border membrane:

emerging structure-function relationships. In Structure and Dynamics

ofChemistry, R Ahlberg and L.-O. SundelGf, eds. (Stockholm: Almquist

Wikesell Intl.), pp. 226-240. Presented at symposium held in Uppsala,

Sept. 22-27, 1977.

Semenza, G. (1979). The mode of anchoring of sucrase-isomaltase to

the small intestinal brush-border membrane and its biosynthetic impli-

cations. In Proceedings of the 12th FEES Meeting, Dresden, 1978, Vol-

ume 53, S. Rapaport and T Schewe, eds. (New York: Pergamon), pp.

21-28.

Semenza, G. (1986). Anchoring and biosynthesis of stalked brush bor-

der membrane proteins: gl ycosidases and peptidases of enterocytes

and of renal tubuli. Ann. Rev. Cell Biol ogy 2, in press.

Semenza, G., and von Balthazar, A.-K. (1974). Steady state kinetics of

rabbit intestinal sucrase: kinetic mechanism, Na+-activation, inhibi-

tion by Tris (hydroxylmethyl)-amino-methane at the glucose subsite,

with an appendix on interactions between enzyme inhibitors: a kinetic

test for simple cases. Eur. J. Biochem. 47, 149-162.

Sjdstrom, H., Noren, O., Christiansen, L., Wacker, H., and Semenza,

G. (1980). A fully active, two active site, single-chain-sucrase-isomalt-

ase from pig small intestine. J. Biol. Chem. 255, 11332-11338.

Sjiistrom, H., Noren, O., Christiansen, L. A., Wacker, H., Spiess, M.,

Bigler-Meier, B., Rickli, E. E., and Semenza, G. (1982). N-terminal se-

quences of pig intestinal sucrase-isomaltase and pro-sucrase-isomalt-

ase. Implications for the biosynthesis and membrane insertion of pro-

sucrase-isomaltase. FEBS Lett. 748, 321-325.

Skovbjerg, H., Noren, 0.. Sjiistrom, H., Danielsen, E. M., and Enevold-

sen, B. S. (1982). Further characterization of intestinal lactase/phlorizinhydrolase. Biochem. Biophys. Acta 707, 89-97.

Skovbjerg, H., Danielsen, E. M., Noren, 0.. and Sjostr6m, H. (1984).

Evidence for biosynthesis of lactase-phlorizin hydrolase as a single-

chain high-molecular-weight precursor. Biochem. Biophys. Acta 798,

247-251.

Sorensen, S. H., Noren, O., Sjostrom, H., and Danielsen, E. M. (1982).

Amphiphillic pig intestinal microvillus maltaselglucoamylase. Struc-

ture and specificity. Eur. J. Biochem. 726, 559-568.

Spies% M., and Lodish, H. F. (1986). An internal signal sequence: the

asialoglycoprotein receptor membrane anchor sequence i s necessary

and sometimes sufficient for membrane insertion. Cell 44, 177-185.

Spies& M., Brunner, J., and Semenza, G. (1982). Hydrophobic label-

ing, isolation and partial characterization of the membranous segment

of sucrase-isomaltase complex. J. Biol. Chem. 257, 2376-2377.

Spiess, M., Schwartz, A. L., and Lodish, H. F. (1985). Sequence of hu-

man asialoglycoprotein receptor cDNA. An internal signal sequence

for membrane insertion. J. Biol. Chem. 260, 1979-1982.

Staden, R. (1982). Automation of the computer handling of gel reading

data produced by the shotgun method of DNA sequencing. Nucl. Acids

Res. 70, 4731-4751.

Strubin, M., Mach, B., and Long, E. 0. (1984). The complete sequence

of the mRNA for the HLA-DR-associated invariant chain reveals a poly-

peptide with an unusual transmembrane polarity. EMB O J. 3.869872.

Tomita, M., and Marchesi, V. T (1975). Amino-acid sequence and

oligosaccharide attachment sites of human erythrocyte glycophorin.

Proc. Natl. Acad. Sci. USA 72, 2964-2968.

Tomita, M., Furthmayr, H., and Marchesi, V T. (1978). Primary structure

of human glycophorin A. Isolation and characterization of peptides and

complete amino acid sequence. Biochemistry 17 4756-4770.

Vieira, J., and Messing, J. (1982a). The pUC plasmids, an M13mp7-

derived system for insertion mutagenesis and sequencing with syn-

thetic universal primers. Gene 79, 259-288.

Vieira, J., and Messing, J. (1982b). A new pair of Ml3 vectors for select-

ing either DNA strand of double-digest restriction fragments. Gene 79,

269-278.

Wacker, H., Jaussi, R., Sonderegger, F!, Dokow, M., Ghersa, P, Hauri,

H. P., Christen, Ph., and Semenza, G. (1981). Cell-free synthesis of the

one-chain precursor of a major intrinsic protein complex of the small-

intestinal brush border membrane (pro-sucrase-isomaltase). FEES

Lett. 736, 329-332.

Wacker, H., Aggeler, R., Kretchmer, N., O’Neill, B., Takesue, Y., and

Semenza, G. (1984). A two-active site one-polypeptide enzyme: t he

isomaltase from sea lion small intestinal brush-border membrane. Its

possible phylogenetic relationship wit h sucrase-isomaltase. J. Biol.

Chem. 259, 4678-4864.

Wickner, W. T.. and Lodish, H. F. (1985). Multiple mechanisms of pro-

tein insertion into and across membranes. Science 230, 400-467.

Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey,

M. L., Goldstein, J. L., and Russell, D. W. (1984). The human LDL

receptor: a cysteine-rich protein with multiple Alu sequences in its

mRNA. Cell 39, 27-38.

Young, R. A., and Davis, R. W. (1983). Efficient isolation of genes by

using antibody probes. Proc. Natl. Acad. Sci. USA 80, 1194-1198.

Young, R. A., Bloom, B. R., Grosskinsky, C. M., Ivanyi, J., Thomas, D.,

and Davis, R. W. (1985). Dissection of mycobacterium tuberculosis an-

tigens usi ng recombinant DNA. Proc. Natl. Acad. Sci. USA 82, 2563-

2567.

Zuker, C., and Lodish, H. F. (1981). Repetitive sequencescotranscribed

with developmentally regulated DictyosteF-lrn discoideum mRN As.

Proc. Natl. Acad. Sci. USA 78, 5386-5390.

86 Hunziker Cell

Documents

Transcript of 86 Hunziker Cell