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).
11766
Dow
nloa
ded
by g
uest
on
Dec
embe
r 14
, 202
0
Proc. Natl. Acad. Sci. USA 92 (1995) 11767
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
300
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
WUZV4Ae33qrbKWYZ, . R
. S AFI.- I . -A m --P6PRTPGVVAAD IIXIIPYPWFIVTDNLPLR GKEVQSA IIH~NI8IZ***Ii$IR*1.M~I 8- P.3SAV
600
ha-MII YBSIPPl IA 5D----TVL l- PI GllTIE - Q
ma-MIX
PDG EVDG5RD Yl lSt FB700
Zr DISC8k ----rFn T V l GXIy0i s2satIuIWR1ksa= V ff s xfl X ^ 1 TVS sX U Y. f zTUlfl RU
hU-MIIx tT8---G&TLFLP SPCb8LCVnSL8LSpLRY8S6--TI-cLWT1 PW----!TSThct-MII s
ma-MIXI LMT--QIQPRRIP TIQOD TAQ SAQS QIRLMQDDNR NIITANLFRILLEKha-Nil T I IBVI4DRRIMQDDNR_ ITLFRILK
Xha-M11 IQYSTAR
m-l-MII RSA STPSLLS SLPCDIBLV'NLRTIX 1BRKGFShot-MIXRA STSl H -UK SLPCDIlRlVNLRITX _ 11.53LKG? 5ha-MHil 8 ---'PCI1DTL:
1000
ma-MIX
ha-IIIhaL-II
S SPMKIST'FRXR W *
-- ---IUE I5T M--- -Q - -- PEBIW8 BAE18POlpt_I81P00ISTFRll*
P ; E F _G*cDWalL-G1100
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.
Biochemistry: Misago et al.
4
I--500
-A-r_-_- -11- L-2 MM- r_.=0
U- I f-,-- - - - . -- - -C
ha-MIin
Dow
nloa
ded
by g
uest
on
Dec
embe
r 14
, 202
0
11768 Biochemistry: Misago et al.
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...........
Proc. NatL Acad. Sci. USA 92 (1995)
Dow
nloa
ded
by g
uest
on
Dec
embe
r 14
, 202
0
Biochemistry: Misago et al. Proc. Nati. Acad. Sci. USA 92 (1995) 11769
-68
ATGAAGCTGRAAAAAGCAGGTGACAGTGTGTGGGGCTGCCATCTTCTGTGTGMRK LIKI Q VT VC G AA I C V
AACTTCCCCCGGAGCCAAATTTCTGTGCTGCAGAACCGCATTGAGCAGCTGQ
CCC
aAA
!CTOL
GACID
OGCOA
TGOO
CAC
ITCII
CAA
Q
PT?T
PT?TF
COGGR
PTC'"
F
CTG(L
TCG(S
CAG
Q
1300G
:AGCS
!ACGT
COGTR
TOGCC
C0OR
lAG
ATO
N
!CTAL
AOCIS
;C
C
Zi
CG
CATTGTCCCCAACTARI TAG GGCA
IS V L QHNR
XCCATGCTGCCCTACTACACGA NL P Y Y T
:CAGAGCTGCAGATGCTCACTP EBL QN L T
C:AGGTGTTTGTGGTGCCCCACQ V FV VP
:CCCGGCGGCGCTTCCTCTGOPR RRFPL W
LCAGCAGGCTOGGTGATGCCBTOGGWV N P
1CAGTOGACCCCTTTGGATAC
A V D PFGY
LGCCTAGAGTTCATGTGGAOOL B F NW R
rGCTGCCAATTTGATTTCAAAIC C Q FPDFRK
PACCGGAAGAAGTCCCAGCTO7YTR IKRKSQL
*.ACT?CT?CAACAGCAGOCCTJD FFHNS R P
'CTGTGCTGAOCGGGGATTTC!.PV LSG D F
OGGGCAGAGGOTTCTGTACAGCIG AB VL Y S
:AGCATCACGATGCCATCACTQ HH8D A IT
?TGCTGOGGOGACAAOGAGACC!V L GDRKH T
.CCAGOQTTTGTOGTCCTATTCP R FV VL
LTCAOCOCACACTGOAGCTCT4I SA WS S
CACCGCACGCTGCCCTCCTCTRT LP S S
IACCGCTACATGvCAGGTCTGGR TN Q VW
TCCAAAGACAAGAGTGGA0CCHIKDIKSGA
LkCTATGAGCACATTCACCAGGT N ST FPT R
LtCATCCATACAGACATCGACART S I QYTS T
CTCACGCTGCACACTGCCCAGLT LH8TA Q
IACAACAAGAGAACCTGCAAC,DHNR R T C
7ACCTGRACGCCCCGGCGCTC,Y LHA PA L
r:GTACGCTCCA=OTGAOGAGR TL Q A
CAAGCAAOOGTAGCCCTGGGCG IKV ALOG
I
GTCV
GMCV
FcN
S
3CMCA
GATD
LOGC
CAGQ
GOcR
MlC
LAC
ETC
:TO
L
NACIY
LAC
XcCA
FTC
V
FTC
TACY
COT
R
3CC
A
(;CcA
R
3CNCA
2AC
D
WOCS
H
NTCC
'CAC
kGAC
MGA
ITCC
AChT
L
:CGIR
ICYCL
.TCC
;OCTA
ACCT
'CACH
'CCAS
ACCT
3COCR
ITCI
L
;TCCS
:AGGR
cYCTL
"FTCF
'cYCL
:ACCT
ICT,L
Q
2GC!
AG
IAT(
FTCI
GCCJA
ACCJT
FOGG
CCTNP
IGCJ
CAT(H
TAT(
A
3iCCiA
FTIN
CTGCL
GAG(B
ATCII
GM(
TTC(
2GCIG
GTOCV
GGT(G
CGC(R
CCTNP
CTA'L
TTC(F
L
:TCCI
GAG
GACID
AATI
ITON
GIACID
GOGG
IAC(H
GTGV
GCCGA
2CTMA
AAG(K
GAC(D
GRAAB
2CG(A
TACY
CTTJL
!CTG4L
TFTTF
CAGAQ
QTC!V
CTC(L
OTAV
ICCClP
CATH
GGCAGCTCGGCCGACTGGGCCCOGGGCGGCGCGGAGGCCGGGCGCTGACGGTGTGTGTGGAGGCCAGT
GiCAGTCA V
;GIGCACE Q
:TGGGTOWV
;CTGCCCL P
:CCAGGC'P G
'TTCTTCIFFr
TCCCACS B
;CCTTACiP T
,TCGGAC'S D
,GOOCCG R
GTCCTCIV L
CAGGCCIQ A
GACCGGD R
'CACGCTIH A
GAGGCTIH A
CCTGAGPHE
CAGGAGQHE
GTCCCTIV P
:CTG;CACL H
'ACTGGCT G
;CCCGATP D
'ACAATCT I
;CCCCGH,P R
TCTAGC88s
CTGCTA,L L
OGCCAGGA R
!TCGGCGS A
GrGCCTOG L
riTTTTGGAGGAGAACCATGAGATTATCAGCCATJL
FTC
V
FTN
TGG
13CCA
TACY
.TO
L
FCC
S
LTC
I
L
ZA0Q
R
A
'GA!R
D
GGCC
'CTL
GGCCG
TGCC
r0GR
CTCL
GAG
ILTG
GAG
D
L
CCI
P
GACD
ITC
I
IAC
mITF
CTC
L
IOC
S
LAC
ITO
V
ZITTF
GAND
:oclR
ITO
V
Xc,P
ErTC
V
COC
R
CTC
L
GAG
CAC
Q
TATY
AAA
COC
R
CAC
Q
ICC
T
FTC
V
CCG(P
lAAC
lAG
W
30kaA
C0C(R
ACAIT
FOCI
C
CCTNP
G3CC
CAT"
PCIN.
S
GFM,V
FTCI
ILGCU.S
FACCY
CAG
Q
lAG
GCAJA
GG00
CTG
L
GAT(D
CGAJR
CTCIL
GCA(A
GTA'V
GAO
MOTOV
ICC!T
TMG
PTML
COT(
GAC
CCTrP
CT?(L
ACT(T
FACIY
300C
GACID
CTCI
L
ITOI
CAMQ
ICYGL
iA0CS
AOCIS
TOO
UAAG
ACC
T
CCA
P
CTCJL
IT?C
;CCC
OGATD
GAC
D
ART?I
0CC
A
ITC"I
TOO
WNGA
CTT'L
TOG
CTG(L
PAT(
CAM
3TGI
MCC
!AT'CI
ICCT
ARGOR
UAAO
!CAG
OGTO
GC
:CT?qL
.GO
R
IAC
D
LkAC
3ACID
LACl
LAO
IAT
D
LCAIT
CT4A
GT
'CTiS
3TCV
.GAR
LCO
T
bC,
.TC
L
'.TGL
bC7
.CTA
L
:AGQ
B
ccc
RAG
RTCI
CAC
0
CTC
L
IAC'D
IAC'D
bGCG
3ATD
I.ToL
ITO
LOC
R
FCC
S
GAC
LOGTS
claT
CAC
-CCAP
I
AGCS
GTGV
FAC
Y
ANT
!CTL
ICC
T
CAC
CCAIP
MlC
EAT
Y
EAT
Y
CGOOR
LtOGR
IAC,
D
:TGL
:CT
P
lGGR
F'TGV
CCI
P
CTC
L
'CTL
V
GAG
CTGL
COT
R
ICC
T
I SH
TTCTTCTCCFF S
TOGAGGCAAIW RQ
TACACAGAGY TE
GTCCAAAAGV QR
ATCGAAGGAIIHO
IGCATGCTGS NL
ITGATGCCCINM P
CCCCGGGCCJPR A
CGATATGACJR Y D
TTTGATGCC'FD A
TACACT?CC(YT S
FACCCACTMYP L
CTTCTGCGCILL R
LCTCOCT?AJTR L
ITCAACTCIN
VHNS
IvTCCOCCTGCV R L
CACGAAGCOHE9 A
G2ATGAGGAODE E
AGGAOCCCCiR S P
FOGACATAT4W TY
CAGvGCCAAC'QAHN
ATCTTGGACII L D
GTCCAAGAT,V QD
CGCTCATTT4R S
AAGGOTT?TTKOGF
TCCTTGACG'SL T
'C
rG
IA
CG
IA
NTCAGCCA1CANTCTGCAAN'I CCTCT
*CACGATCCCACCCGACACCAGAATGGTGGGGH
IATCI
!ATC
G
;CAGQ
;AGA,R
LCAC,H
ATT
ITTC
ATC
AAO
CTG?L
COOR
TCT4S
OTM
CCA(
nTYF
.CCC
"CATH
F
R
'AOCIS
!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
Dow
nloa
ded
by g
uest
on
Dec
embe
r 14
, 202
0
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.
Dow
nloa
ded
by g
uest
on
Dec
embe
r 14
, 202
0
Top Related