Proc. Natl. Acad. Sci. USAVol. 92, pp. 11766-11770, December 1995Biochemistry

Molecular cloning and expression of cDNAs encoding humana-mannosidase II and a previously unrecognized a-mannosidaselix isozymeMASAHIRO MISAGO*t, YUNG-FENG LIAOt, SHINICHI KUDO*, SUMIYA ETOt, MARIE-GENEVIEVE MATTEI§,KELLEY W. MOREMENt, AND MICHIKO N. FUKUDA*¶*Glycobiology Program, La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA 92037; iComplex Carbohydrate Research Center,Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602; §Physiopathologie Chromosomique, Institut National de la Santeet de la Recherche M6dicale, Unite 406, Genetique Medicale et Development, 13385 Marseille, Cedex, France; and tFirst Department of Internal Medicine,School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807, Japan

Communicated by Helen M. Ranney, Alliance Pharmaceutical Corp., San Diego, CA, July 28, 1995 (received for review June 9, 1995)

ABSTRACT Golgi a-mannosidase II (a-Mll) is an en-zyme involved in the processing of N-linked glycans. Using apreviously isolated murine cDNA clone as a probe, we haveisolated cDNA clones encompassing the human a-MII cDNAopen reading frame and initiated isolation of human genomicclones. During the isolation of genomic clones, genes relatedto that encoding a-MIl were isolated. One such gene wasfound to encode an isozyme, designated acMIIx. A 5-kb cDNAclone encoding a-MIIX was then isolated from a humanmelanoma cDNA library. However, comparison betweena-MIIX and a-MIl cDNAs suggested that the cloned cDNAencodes a truncated polypeptide with 796 amino acid residues,while a-MI consists of 1144 amino acid residues. To reeval-uate the sequence of a-MIIx cDNA, polymerase chain reaction(PCR) was-performed with lymphocyte mRNAs. Comparisonof the sequence of PCR products with the a-MIIX genomicsequence revealed that alternative splicing of the a-MIIXtranscript can result in an additional transcript encoding a1139-amino acid polypeptide. Northern analysis showed tran-scription of a-MIIX in various tissues, suggesting that thea-MIIX gene is a housekeeping gene. COS cells transfectedwith c-MIIX cDNA containing the full-length open readingframe showed an increase of a-mannosidase activity. Theca-MIIX gene was mapped to human chromosome 15q25,whereas the a-MIl gene was mapped to 5q21-22.

a-Mannosidase (a-M) activities are involved in both biosyn-thesis and catabolism of N-linked glycans (1, 2). These enzymeactivities are present in cells ranging from yeast to human.There are different forms of a-Ms: lysosomal a-Ms are solubleand involved in degradation of N-glycans, endoplasmic retic-ulum (ER) and Golgi a-Ms are involved in processing of newlysynthesized N-glycans, and cytoplasmic a-Ms may be involvedin degradation of dolichol intermediates that are not neededfor protein glycosylation or oligosaccharides derived fromglycoprotein turnover in the ER (1).. Substrate specificities ofthese a-Ms differ from each other, and Golgi a-MIl specif-ically hydrolyzes two peripheral mannosyl residues fromManal1-6(Manal --3)Manal ->6(GlcNAcf31--2Mana1 ->3)[Man/31 ->4GlcNAc,lB --4GlcNAc31 ->]asparagine struc-ture. Several a-Ms have been cloned to date. These includeGolgi a-MIT (3, 4), ER/cytosolic a-MI (5), two isozymes ofGolgi a-MI (6-8), lysosomal a-M (9), Dictyostelium a-M(10), and yeast a-M (11) (for a recent review, see ref. 2).a-MII is a type II membrane protein mainly residing in the

medial Golgi cisternae, while its localization is cell-type de-pendent (12, 13). A genetic defect of a-MIT in humans causescongenital dyserythropoietic anemia type II or HEMPAS

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

(hereditary erythroblastic multinuclearity with positive acidi-fied serum lysis test) (14). Thus, the reduction of a-MIT activityresults in a failure of polylactosaminoglycan formation inerythrocyte membrane proteins, leading to clustering of mem-brane proteins and formation of unstable erythrocytes (14-17). Since a-MIT is normally expressed in a variety of cells intissues, the HEMPAS defect is not restricted to erythroid cells(18, 19). However, there are cells and tissues that are notaffected by HEMPAS genetic defect. This predicts the exis-tence of one or more tissue-type-specific a-MIT isozymes (16,17) that compensate for the a-MIT defect in certain cell types.The present report describes the cloning of human a-MIT andthe genomic and cDNA cloning of an a-MIT isozyme desig-nated a-MITx and their sequences.l

MATERIALS AND METHODSMaterials. Full-length mouse a-MIT cDNA clones and

cDNA clones containing the partial human a-MIT open read-ing frame have been isolated (3). Additional human a-MITcDNA clones were isolated from a human liver cDNA libraryin the Uni-ZAP XR vector (Stratagene). A human cosmidgenomic library constructed in pWE15 cosmid vector (Strat-agene) was provided by T. Sato, La Jolla Cancer ResearchFoundation. A melanoma cDNA library constructed fromhuman melanoma cell line SK-Mel-28 (ATCC HTB 72) in thepcDNAI vector was purchased from Invitrogen.

Isolation of a-MIT cDNA Clones. The human liver cDNAlibrary (-1 X 106 plaques) was plated on XL-1-Blue host cellsand screened by plaque hybridization using standard proce-dures (20). The probe used for the library screening was a32P-labeled 578-bp EcoRI restriction fragment derived fromthe 3' end of the previously isolated human a-MIT clone, HM-1(3). Clones were excised from the Uni-ZAP vector as insertsin Bluescript II SK(-) and were sequenced. A single clone,designated HM-4, was completely sequenced and found to be2825 bp long, to overlap the 3' end of the HM-1 clone startingat base pair position 810 of HM-1, and to contain 163 bp of 3'untranslated sequence followed by a poly(A) tail. The full openreading frame of human a-MII cDNA was assembled fromthose two clones by ligating the 5' 1258-bp HindIlI fragmentof HM-1 with the 2383-bp HindIlI fragment of HM-4.

Screening of the Human Genomic Library. About 1 x 106colonies of the cosmid library were hybridized with a 32plabeled 1.4-kb EcoRI fragment of human a-MIT cDNA as a

Abbreviations: a-M, a-mannosidase; a-MI and a-MII, a-Ms I and II;HEMPAS, hereditary erythroblastic multinuclearity with positiveacidified serum lysis test.ITo whom reprint requests should be addressed.'The sequences reported in this paper have been deposited in theGenBank data base (accession nos. D55649 and L28821 for a-MIIxand U31520 for a-MI).

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probe (20). Two positive clones with an insert size of about 40kb were isolated and were designated clone 7 and clone 14.

Screening of a Human Melanome cDNA Library for a-MIlxClones. A human melanoma cDNA library in the pcDNAIvector was screened with a 69-bp PCR amplimer probe derivedfrom genomic aMlIx clone 7 (see Fig. 2). A clone with an insertsize of 5 kb was isolated and sequenced (pcDNA-IMX).

Partial cDNAAmplification by Reverse Transcription (RT)-PCR. Poly(A)+ RNAs were isolated from normal humanlymphocytes, and cDNAs were synthesized by using a First-Strand cDNA synthesis kit (Pharmacia). PCR was performedwith the following oligonucleotide primers: 5'-TTTCTTCTC-CTATGCGGACCG (nucleotides 1476-1496 in Fig. 3) and5'-CCAGCTCCTTGTTGACGTAGTC (nucleotides 2652-2631 in Fig. 3). The PCR products were subcloned intoBluescript vector and designated pBS-LymRTPCR. Genomicclone 14 was also subjected to PCR with the primers 5'-GG-CCTGGGCGTGCAGCTA (nucleotides 2140-2160 in Fig. 3)and 5'-AGACAAGGACCTGCATGTCCA (nucleotides2404-2384 in Fig. 3). A PCR product of about 500-bp wassubcloned into pBluescript and named pBS-genomePCR.

Sequencing. Nucleotide sequencing of the cDNA andgenomic clones encoding a-MIlx was performed by dideoxy-termination methods (21) using a Sequenase kit (United StatesBiochemical).

Construction of a Mammalian Expression Vector Harboringa-MITP cDNA. Because the subclone pcDNAI-MX, isolated from

m-MIXIha-MIIha-MI4J

mat-NIIha-MIIha-MIX4

ma-MII

the melanoma cDNA library encoded a truncated a-MIIx prod-uct, a cDNA encompassing the full coding region of a-MIlxcDNA was obtained by replacing the Cpo I fragment (1095 bp)of melanoma cDNA with a Cpo I fragment (1070 bp) of pBS-LymRTPCR. The resulting clone was named pcDNAI-MIIx. Thelarge EcoRI fragment from pcDNAI-MIIx containing the entirea-MITx cDNA open reading frame was excised and ligated intothe EcoRI site of a mammalian expression vector, pXM (4).

RESULTSIsolation of Human a-MII cDNA Clones. Partial cDNA

clones containing the 5' end of the human a-MIl open readingframe have been previously isolated (4). Clones containing theremainder of the 3' end of the open reading frame wereisolated by plaque hybridization with a 578-bp EcoRI restric-tion fragment as a radiolabeled probe. The longest of theclones overlapped with the previous clones and extendeddownstream through the end of the open reading frame and163 bp into the 3' untranslated region before terminating in apoly(A) tract. A full-length human a-MIT cDNA open readingframe was assembled from these clones. The open readingframe encompassed by this construct encoded a product thatwas 1144 amino acids in length and 80% identical to theproduct of the cDNA sequence of murine a-MIT (Fig. 1),including a 97% identity over the NH2-terminal 87 amino acids.

MU8QTF8IFCWIF8LYLMLDGLY * G8xIDRPT4imiisNnwsvimLszzsU ^N^ Q

F wS ]~~~~~~ PCLFAQ8GI8QV*LaDPVGQFDBSBNDPGKTNDYPRDKTQYIFNNM

100

VKK ISY_M_33 II(=QL#UVPSW[PGSTAhot-MII VIKLKD 8RNt81CI8MI IDI IlSE6IlIVGVWDST lYIIDOLIE!GElPG8GAADPFGHSPTMAYha-MIIX ti~- ~ ~

200

ma-MIIha-MIXha-MIl

ma-MIXha-MIXhot-M1I

m-MIXha-MIXht-4IIe

mma-MIIha-MIXha-MIIl

LXI 4dBLIQGvuYA --R---_FF_RL ILCENMPFYSYDIPH.TCGPDPKICCQFD P A1

IX SHhI WHYJHFA TL3FFWRC lt ILCHMMPFYSYDIPSTCGPDPKICCQFD RL^1h AtIORvDyAIKRVB FaITBrEE&eIrF D3CnMMPFYSYERI*PTCGPDPKICCOF_D

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a RKKSKLFRYKREVLaPLGDD G'DTYQIQFG D A MLSGDFFTYADRa E Q RRS DFRDLQw NYQ1I~LFDYMN81T^DIQFg% GpFFFYAR

+400

DDIYG SRPYKRNDRFD RIIFW D DIAS Y ^DDYWGYFT GYR8RYRIRILILS I L88X DAIILis

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3DA P IG. 1. .omparison otamino acid sequences betweenmouse and human a-MIIs andhuman a-MIlx. The boxes indi-cate identical residues betweenthe lines of sequence.

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Forward primer Reverse primer

Genomic clone:...ATGAAGCTGAAAAAGCAGGTGACAGTGTGTGGGGCTGCCATCTTCTGTGTGGCAGTCTTCTCGCTCTACCTCATGCTGGACCGAGTGCAACACGAT...

a-NII cDNA :...ATGAAGTTAAGCCGCCAGTTCACCGTGTTCGGCAGTGCGATCTTCTGTGTGGTGATTTTCTCGCTCTkCCTGATGCTGGACCGGGGTCACTTAGAC...1

FIG. 2. Partial nucleotide sequence of genomic clone 7 and corresponding cDNA sequence of human a-MIT. DNA sequence obtained froma subclone of the 2.4-kb fragment. Primers were designed of positions indicated by the arrows for amplification of the 69-bp probe used in cDNAlibrary screening.

Hydropathy analysis on murine (4) and human a-MIT andprotease studies on the murine enzyme (22) indicate that a-MITfrom both species is a type II transmembrane protein with anNH2-terminal 5-amino acid cytoplasmic tail and a single trans-membrane domain within this highly conserved sequence region.Expression in COS cells (4) resulted in the overexpression ofa-MIT activity toward the synthetic substrate, p-nitrophenyl a-D-mannoside, and the appearance of anti-a-MII antibody cross-reactive material in a juxtanuclear membrane array consistentwith the Golgi complex (data not shown).

Screening of a Human Genomic and cDNA Libraries andIdentification of a-Mllx Gene. A human genomic libraryconstructed in cosmid vector pWE15 was screened using32P-labeled human a-MIT cDNA as a probe. Two positiveclones, each with an insert size about 40-kb, were isolated.These two clones were designated clone 7 and 14, digested withEcoRI, and then analyzed by Southern blot hybridization using32P-labeled a-MIT cDNA as a probe. EcoRI fragments of2.4-kb and 4.0-kb from each clone were found to hybridize withthe probe and were subcloned into pBluescript. The nucleotidesequence of these subclones showed that the sequences aresimilar but not identical to a-MIT DNA (Fig. 2), suggesting thatclone 7 encodes a new gene related to the a-MIT gene. Theproduct of this new gene was named a-MIlx. A humanmelanoma cDNA library was then screened by using a short(69-bp) DNA fragment as a probe (Fig. 2), and one positiveclone with an insert size of 5-kb was obtained. The nucleotidesequence of the cDNA clone had an open reading frameencoding 796 amino acid residues (Fig. 3).To determine if a different cDNA sequence encoding

a-MITx is present in different cells, RT-PCR was performedwith poly(A)+ RNAs isolated from normal human lympho-cytes. PCR primers were designed so that the PCR products tobe sequenced would cover the area between nucleotides 1476and 2652, which is the region containing the stop codon at2389. The sequences of lymphocyte PCR products revealedthat there is an alternative a-MITx cDNA sequence that has a25-bp deletion when compared with the sequence of thea-MITx cDNA clone isolated from the melanoma library (seeFig. 3). When the sequence obtained by PCR replaces thecorresponding sequence in the melanoma cDNA clone, theresulting product of translation has 1139 amino acid residues

(Fig. 4), which is comparable to the size of human a-MII (1144amino acids). Hereafter, the cDNA product of 1139 amino acidresidues will be referred to as a-MITx.A hydropathy plot (23) of the a-Mllx polypeptide showed an

uncleavable membrane-spanning domain near the NH2 terminus,suggesting that a-MITP is a type II membrane protein. Alignmentof peptide sequences shows a close similarity between humana-MIIx and human and mouse a-MIT, respectively (Fig. 1).

Alternative Splicing of a-MIIX Gene. The sequences ob-tained from a-MITx genomic clone 14 revealed that both cDNAsequences obtained from melanoma and lymphocyte cells arepresent in genomic clones. Melanoma cDNA contains an extra25 nucleotides (see highlighted letters in Fig. 3) after residue2347, leading to a frame shift with a stop codon at 2389. Onthe other hand, lymphocyte a-MIlx mRNA spliced out anadditional 25-bp, yielding an open reading frame extending tonucleotide 3417, thus encoding a polypeptide with 1139 aminoacid residues. RT-PCR analysis showed that these two formsof a-MITx transcripts are present in various cell types (data notshown), suggesting that the a-MITx gene generally producestwo alternatively spliced mRNAs in normal human cells.

Expression of ai-MIIx in Various Human Tissues. RNAhybridization (Northern) analysis of a-MITx (Fig. 5 Left)showed that a-MITx mRNA is seen as a single band at 5 kb invarious types of adult tissues. Northern analysis of a-MIl anda-MITx showed that both enzymes are strongly expressed in theplacenta and kidney; in heart, brain, and skeletal muscles,expression of a-MITx is stronger than that of a-MIT. Expressionof these enzymes is very weak in lung.Enzyme Activity of a-MIIx. COS-1 cells were transfected

with plasmid constructs containing human a-MII or a-MITxcoding regions and assayed for overexpression of a-mannosi-dase activity as described (4). Cell lysates prepared from theCOS-1 cells transfected with a-MITx cDNA exhibited a 1.6-foldincrease of a-mannosidase activity compared with those ofcontrols (Table 1). These results indicate that a-MITx polypep-tide with 1139 amino acids is catalytically active and canhydrolyze p-nitrophenyl a-D-mannoside.Chromosome Localization. a-MITx gene and a-MIT gene

were mapped to human chromosomes 15q25 and 5q21-22,respectively (Fig. 6).

[pBS-genomePCR][intron] 230bp

..... GGGCTCCTCAAGgtaaa........ acatgctggtgtggagcaa GCATCCGAAGGGTGGATQRGGAGCkC.

[pCDNML-NZ]2347 2389

.... 1IAIc7 A UCCGTiAGGAGTGGATGRG....G L L K G S G L C F L A HB P K G G end

783 796

[pBS-Ly RTPCR]2347

.....GGGCTCCTCACAT1GAAOGGG GCrCAGCAGCAC....GLLK S I R R V D EEB Q Q

783

3418

rGTGGACATGCAGGTCCTT..............CGCTTGGGTTAGGGCTTCV D M Q V L ...............R L G end

1139

FIG. 3. Two alternative splicings of the genomic DNA encoding a-MITx, resulting in either short (796 amino acid residues) or long (1139 aminoacid residues) a-MIIx polypeptide. The sequence of 25-bp nucleotides (highlighted) is present in genomic clone 14 (top line). The sequence thatbecomes a part of the exon by an alternative splicing is highlighted (middle line).

'A --- ^^.^^.;GACKACGA...........

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!CATH

!GAC,D

'TTASL

D

lAGGK

FCCI

TI

rNCGGA

LACA(

T

2CCC

rACAI

:CCN"

IATNIDI

LI

:ACGH

:0CClRI

;CC(!A3

:CTCIP

GAGCI

TOC7CI

CCC7PI

TACCIY

COGGR

CACTH

CCTCP

FWOCCI

CTGZL

P

IAC!D

CCG(P

GACJD

CAN

GCA(A

FOGG

AGA(R

2AGC

CAGCQ

PLAG)K

FTCl

2TCI

GACCID

UTGC

L

CAG(

FCAM

TC(

CT=L

CTGiL

T0CT

G

TACIY

T R

s V

CAGGAC'Q D

ATCTCCII S

CACATCH I

IGTCCGA&V R

CTGGAG

LHBIGTGCAC!VHBPATGAC4Y D

GCCAAC'A N

GAGTGG(1WNAGGACA(A T

TACAAG)

ICCCTC(T L

AACCTGi

GCCCTC(A L

CGTGTCCR V

GGCCTGCG L

CGTGTrcR V

CAGGTO

2TGGTCA'

W T

ATGCCAI

ATGCAGN Q

ACCAGC'T S

2CTTCC!A S

CTGGAOLIBCCTCTOP L

H

;CTCL

TGOCC

'TAC,Y

ICTCL

kAGOR

AkGAR

TAT4Y

:GTCIV

!GToV

OAT4D

IGGCG

AGC!S

~CTGL

AAG(K

CCA(P

CTT!L

OGOCG

ATEII

;GACD

LCTGL

T-CC,S

OGTCV

OATD

TACY

TCA,S

0GCCA

0GCCIA

Q

GAG

CAG

r2ACD

PLAT

CTCL

PLATH

A

XCC

3CA,A

3CCA

3TOV

NTAIL

ICOT

:AGQlAGH

'COS

GACD

ITC

LAGI

LTCN

rIACD

rCCAP

CTCL

RAGITCCS

H

CTCL

CCG

AGC

FTC

CTNL

ITCI

C-AN

rlAO

CAC

GAO

GAC,D

lAAM

WTC

R

lAG

ITL

TClS

CAC0

3CCA

ACTT

2CCA

AACH

TCCS

cccP

AACH

CCGP

0

CAMT

2GCTIA

CAC(H

ATGON

GG0k0

GGTN0

lAGII

ICCIT

1004R

TTCIF

CCAIP

COACR

2cINA

PLTCUI

PkCGCT

rlAGCB

2CAG

CTML

ACCOT

COGGR

CTC(L

PTGTC

PTG(L

TCCJS

G

0GCCA

-TTOL

!GACD

GTGV

AACH

GCAA

lAGI

POTC

GCAA

TTCF

2000

2TCV

COGR

ITTI

GTGV

GGTG

CTAL

ICCT

CTTL

FTCTS

TCAS

IATCI

GOC0

~CTCL

GACD

IACH

KG

:G

3c

Ik

CG

(CANCC GTTCGCN TTATCACGCGCGT WCTACGCCTGAOOC417 I K A CCGCCTIAGGC GGC AGAGU

KCGCTTN CNTACG NCACTTCC(GCACKCTCGGGCTT CGAGCTTCGTTGCAGCCAGA(GGTCCATI

A(TCCAGI TTTGCTTTCAACGGCACTCTGGAGCATTGCCCTCGTTTGTCCTGTACAAAGGACAGGTGC CCAC

KCGCTN GGTCCTTCGNCGGTCTCCTTGG (GGGhTATCTN 1 C NC N ATTCGCCTCACAC TTA(IkCAT

hGCAC CGGGGAAGTCGAACCT NAGTAGTCCGTTGTTCTCGGCC'(NTCCCTTTATTACTC TGACGGAAGTG

AATGGGCCTCCACTAGTCACCTGGC CCAAGGGTTGTGGGCATGGCA CTTTCCCCTGCACTGACAGGCATAT

IGAGCCATA CTGTNTGGNACAC CCTACATTGCCCTGGCCCTCCAGGAGAATGTAGCCAGCTC CAGCGCCGT

ATCAGTGTCCCCCKTTTOCCATACCI ACTGAAGGGTCGAvTGCCGTGCATTCCCTCGNTGTCCTTCTNCGGGAGG

;CCCC;CCTGAGTCGCCAACGGGCCTGCCCIGGGCCGAGCTCAGTTTTGNGTCCTGCGCCTGCGCCGGCGTCTGCAGT

;CGGCCGGTGGCCCCAGTGCCCCCTTGGCTCN CTC GGCGC:CTTCGC4CCi CGAGGTTCTTCGTTCTT CCTGCC

'GCTCCCTCGAACGTCAGAGTCGTTTCGCCTATGGNCTAAhCACCiGGK IG GGCCGGAG(GTGAATCGGC

NTTGCCCGAGTCAN 7ANG CCACTTCCGCC INGGAGCTAGCCIAG CCCCGGTGGGTATGAGCCT

TCCAA ITKC TCATA:CCTTACAG :7CGCTTCATTCC:4GTGA CNNGCCTGC NCACCTGG :1UCGGATAG

AGGTGCCGACTCTAA ACGCGCA GGAGCCCACGTTCGAGACTCCCK AGCCATTTACCATCAG:4GGCTT

C IGhLCCA A CGCTAGTGAATCCAGCTGTGCCACTC1GTGCACG1KG CTGCGGGCGTCA1GUTGCUACGG

1GCC GGCCAGCCCAGG1ACTCCGTTCGCTCTCTG iCGCGACGGGCATjGTCAAI TGCATCACAGTCCATCCC

AGCACGhCCCTTNCThCGCCGCCTGCCGCTTAC AGGTCGTCCGCCTG GGTCTTACCCGCTCTCACTGCCATGT7

:CCCGTCACTCGACCCC4 T KAGA4CCTCCTGCUGG CGAC NCTACACI AGTTTGCGCGCTGAGCAGUC:G

AACTGCCCACAACCAGC NTGCTGGACTTTCTGCTGTNGTTCTCACACTCTTGCGTACTTCCTTGCTCCGTC

-1

12040

24080

360120

480160

600200

720240

840280

960320

1080360

1200400

1320440

1440480

1560520

1680560

1800600

1920640

2040680

2160720

2280760

2400800

2520840

2640880

2760920

2880960

30001000

31201040

32401080

33601120

H T D VYL E P N I ATFPR LRLGOGod 1139

ATTGRCAhCCAACGGTATCCATCCGhCTGCTCCAGRATGTRCACCTGGCTCGCCCCATTTCTTTTATGCGCTGTGTGTTTGGCGAACCACAAC36000AGTGKTGGGAAATAGGGCGhCGCCAGThGATCAGGGGhGRAGGCCTTGGTCAGGTGGGCAGTCCAGGCTCTC --3720

C,CACCCKTC CCAGCATCAGGGTCACTGTGGCARCAGCAGGCTCTAGGG ~~~~~~~3840

GRATCc CCAGhGTTAGCAGTGGGGATGGRGCAOTAGAAGGAATC ~~~~~~~~~3960

ACTGTTCCCTAGRGCTGRGGCTCGCGCTTCTGGRTGCTTGCATAAGGCCCCTGCCTCATCATTGCTACAGCTGAGRAGGCGGRAGACCGCTG40808

ATTGCATOCCGAGTCAAGGTACTCACGrACAAACCCTGGAAATGGCACTGGCTATTACGATTGTGGATGTGCAGGGTTGGGTCCTCACCTC42000GRATTTCTTTGC.r.lc TGATGCTCTGTCCCAAGGCTCTTCTTTTATGCAGGGGTGGGhTTGGCCTACCTCCAGACT43202

ATGTCTTGGCACTTRGRAGCGTCAGACCCGCCAGCGGCAKCCTGhACCTACCTTAGCTACTGCGAGTATTAGTCCTGCACGGGGCTAGGGCT44404AGhGATAGRGGGGCGTATGCTTGCAGAGCTTCTTTAGCTCAATAATGGGAGRTTTAGATGCTCATAATGCGAAAGGTTTTGTAAATTAAAAC45606TCCCTATTAAATCTTGTAATTAGTCGGRAGRACACCTAACTAGTAKGGTC PCAAAACAACGTGKGGCTTCTAAGGTCAACGCACAGCATC46808

ATTAAGTArmAACTGCCAGGGAAGCATTTTCACAGGTCCGGCCTACAACCCTGAACAGCCGATAGGACATAGGAGAATATATTOCATTTTTTA48000ThMCTGCCTACCTGTAAAA 4824

FIG. 4. Nucleotide sequence of a-Mllx cDNA and deduced amino acid sequence.

DISCUSSION two highly related cDNAs encoding enzymes involved in the

Recetlyit hsbcomeevient hattrimingstep inthe cleavage of a(1,2)-mannosyl units have recently been cloned

(6, 8). We have also identified clones encoding a previouslymaturation of N-glycans (24) that were previously attributed to undisclosed a-MII-related gene. A 69-bp probe that was

a single processing hydrolase can potentially be catalyzed by distinctive to this a-MIT-related sequence was successfullymore than one isozyme in mammalian tissues (2). In particular, used to isolate a full-length cDNA clone (Fig. 4).

H F

AACGCAH A

GOGGGCGOG

TGGGATW D

TjLCTAAGSRK

GGGCACG Q

ACCCCCT P

CACTTTHFp

GGCCCAMO P

OCCCTOA L

AACTAC,W Y

GCCCGGA R

CTOGGMLBE

CGCACAR T

CATGCA,H A

ATCCAGI Q

CAGCCCQOP

CAGCTGQ L

AGCGACS D

GTCTATV Y

TCTCACSOQ

ACAAGGT R

CAGGACQ D

CTAGGC,L G

AGCCAC,88B

TTCCAC,F H

TTCAAF H

P

GAG

CGGR

GCTA

CTCL

CTCL

COCR

2CTA

GATD

CTNL

CAA

CCN

A

FTL

(;CcA

CTCL

!CTL

lTT

GCC0

lOCR

lOCS

2C1CA

CAA0

CYCL

CTCL

OTCC

R S

2GCCCGAO P

GGTCAGG Q

GAAGACE D

CAGGAG05B

2AGATT(H I

TCTGGCISOG

GCCACC(A T

CCCAAGPRx

CTGG&C(L D

CGGCTCIR L

CCAGGGMP G

CACCTGCH L

300CTCIGOLCACTATNH Y

GATTCCID S

GCCGTGCA V

CTGGAT4L D

2CCCTCA L

ACCCGTT R

TGGTTOW L

TGGCCC.W P

CAGRAAQ R

200CTCJGOL

ACCTCCJT S

CTCAACLHN

ACCACAUT T

ic-GMrvcxn

aInTCGTzc.1IxCTNC7r-A3cICxX.ACrvcraG:9I1TGci3G:GxGGhG

ICGCcc:I:A:TVGTTGPGxICCArG3TXxGGG".PGUGGcc

;CCArA:AXLC3hxC7GT7-A.1IMCArpACTNK.CA24::TXTGTG

LAGGGGuLCkG3iTGr-A::Axn07CA:G.1II1GGPM3G'C'xacroc

XTGPCVXXtcr-ADCIC9LiTGCTn.m:AOG14PAGK.OGGc

CA2A.1ILGrcvCAr-AICKroc3cICx:ACAa,::A11CTcc

rAZIC'C'IIC1711X.hT

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Proc. Natl. Acad. Sci. USA 92 (1995)

h(-1P b 0&441

9.5-7.5-4.4-

2.4-

1.35-

2.4-

1.35' w1.35-

,eW,0404ffi '0 sS4,S N

5

15

9.5-7.5-

4.4-

2.4-

1.35-

2.4-

1.35-

FIG. 5. Expression of a-MII and a-MITx mRNA in normal humantissues. (Upper) The filter (multiple tissue Northern blots, Clontech)was hybridized with either human a-MITx cDNA probe (Upper Left) orhuman a-MII cDNA probe (Upper Right). (Lower) Each filter wasstripped of its probe and rehybridized with ,3-actin cDNA probe. Thenumbers show the position of standard size markers in kb.

The present study shows that expression of a-MITx is influ-enced by alternative splicing. As described above, a-MITxmRNA is spliced differently in the coding region, and suchalternative splicing results in either active or inactive enzyme.PCR analysis of several tissues showed coexistence of twoalternatively spliced mRNAs, suggesting that cells regularlyproduce both active and inactive a-MITx. It would therefore beinteresting to know whether any factor that affects the modeof splicing also controls the level of N-glycan processing in vivo.We have predicted (16, 17) the existence of a fetal type

a-MIT isozyme. Such a hypothesis stemmed from an analysisof HEMPAS disease; HEMPAS patients do not show anobvious abnormality at the time of birth (18). This fact isintriguing, because N-glycans present in plasma membranesare involved in cell-to-cell interactions, and therefore may playimportant roles during embryonic development. In fact,"knock-out" of the N-acetylglucosaminyltransferase-I gene inmice resulted in embryonic lethality, presumably because theconversion of high mannose to complex type N-glycans isimpaired (25). It is therefore possible that a fetal-type isozymeof a-MIT is expressed in the human embryo, thus avoiding thegenetic defect of a-MII in HEMPAS. Further analysis ofa-MIT-related genes will define an apparently developmentaland tissue-specific manifestation of HEMPAS disease.

Table 1. a-Mannosidase activity in COS-1 cells transfected witha-MI- and a-Mllx-related cDNAs. Numbers presented are theaverage of quadruplicate analysis

a-Mannosidase activity,% of control

(mean ± SEM)

pXM 1.0pXM-murine MII 2.5 ± 0.3pXM-MTTx-sense 1.6 + 0.2pXM-MTTx-antisense 1.0 ± 0.1

3--Q- P 1112--

7.6

is :

2-

2- 3-

5 - - * -- -- *0006_ _ *@-@@@@@

0S

FIG. 6. Distribution of labeled sites on chromosome 15 for a-MIIx(Left) and chromosome S for a-MII (Right). Idiograms for the respec-tive chromosomes were prepared as described (26).

The authors thank Dr. Takaaki Sato for his help in screening ahuman genomic library, Cherylance Ponder for screening the humanliver cDNA library, and Phuong Thai for her technical assistance. Thiswork was supported by National Institutes of Health Research GrantsGM47533 and RR05351 to K.W.M. and DK37016 to M.N.F.

1. Daniel, P. F., Winchester, B. & Warren, C. D. (1994) Glycobiology 4,551-566.

2. Moremen, K. W., Trimble, R. B. & Herscovics, A. (1994) Glycobiol-ogy 4, 113-125.

3. Moremen, K. W. (1989) Proc. Natl. Acad. Sci. USA 86, 5276-5280.4. Moremen, K. W. & Robbins, P. W. (1991)J. Cell Biol. 115,1521-1534.5. Bischoff, J., Moremen, K. W. & Lodish, H. F. (1990) J. Biol. Chem.

265, 17110-17117.6. Lal, A., Forsee, T., Scutzbach, J., Neame, P. & Moremen, K. W.

(1994) J. Biol. Chem. 269, 9872-9881.7. Bause, E., Bieberich, E., Rolfs, A., Volker, C. & Scmidt, B. (1993) Eur.

J. Biochem. 217, 535-540.8. Herscovics, A., Schneikert, J., Athanassiadis, A. & Moremen, K. W.

(1994) J. Biol. Chem. 269, 9864-9871.9. Nebes, V. L. & Schmidt, M. C. (1994) Biochem. Biophys. Res. Com-

mun. 200, 239-245.10. Schatzle, J., Bush, J. & Cardelli, J. (1992) J. Biol. Chem. 267,

4000-4007.11. Camirand, A., Heysen, A., Grondin, B. & Herscovics, A. (1991) J.

Biol. Chem. 266, 15120-15127.12. Moremen, K. W. & Touster, 0. (1985) J. Biol. Chem. 260, 6654-6662.13. Valasco, A., Hendricks, L., Moremen, K. W., Tulsiani, D. R. P.,

Touster, 0. & Farquhar, M. G. (1993) J. Cell Biol. 122, 39-51.14. Fukuda, M. N., Masri, K. A., Dell, A., Luzatto, L. & Moremen, K. W.

(1990) Proc. Natl. Acad. Sci. USA 87, 7443-7447.15. Fukuda, M. N., Klier, G. & Scartezzini, P. (1986) Blood 68, 521-529.16. Fukuda, M. N. (1993) Bailliere's Clinical Haematology, edited by

Tanner, M. J. A. & Anstee, D. J. (Bailliere Tindall, London), pp.493-511.

17. Fukuda, M. N. (1990) Glycobiology 1, 9-15.18. Crookston, J. H., Crookston, M. C., Burnie, K. L., Francombe, W. H.,

Dacie, J. V., Davis, J. A. & Lewis, S. M. (1969) Br. J. Haematol. 17,11-26.

19. Fukuda, M. N., Gaetani, G. F., Izzo, P., Scartezzini, P. & Dell, A.(1992) Br. J. Haematol. 82, 745-752.

20. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview,NY), Vol. 2.

21. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci.USA 74, 5463-5467.

22. Moremen, K. W., Touster, 0. & Robbins, P. W. (1991) J. Biol. Chem.266, 16876-16885.

23. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132.24. Kornfeld, R. & Kornfeld, S. (1985)Annu. Rev. Biochem. 54, 631-664.25. Metzler, M., Gertz, A., Sarkar, M., Schachter, H., Schrader, J. W. &

Marth, J. D. (1994) EMBO J. 13, 2056-2065.26. Nguyen, C., Mattei, M.-G., Mattei, J.-F., Santoni, C., Goridis, C. &

Jordan, B. R. (1986) J. Cell Biol. 102, 711-715.

11770 Biochemistry: Misago et al.

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