i 70
BBAPRO
B,,~ h~mi¢ a e~ B~,Jph~,,tt'u .4t ra. 1037 ( I qqO) 170-177 El,,¢v~o"
33534
Molecular c!oning and expression of a c D N A encoding the membrane-associated rat intestinal alkaline phosphatase M a r k L o w e ~, A r n o l d W. S t r a u s s -"~, R u t h A i p e r s ~ S h a k u n t l a S e e t h a r a m a n d D a v i d H. A l p e r s t Departotenls of t Medwme. : Ped~utr,'s
und
~ B~olog~¢al
(.'hem~tr)'. 14"ash~ngto~r L'nwer~t~ Med~(al ~ k o o ~ S t l~u~s. M O I U S A )
(Received ~ Jul} i98~))
Key words: Alkaline phosphala~: Phosphaudylnno~ttol
Rat intestinal alkaline phosphatase (lAP) has been pmilied and Woteolytic fragments sequenced. A eDNA h'larmT was constructed from duodenal poly(A)* RNA and screened for lAP positive clones by a f~l-length eDNA ¢kmc--eocot~l~ human lAP. A full length rat LAP ~ (2237 bp) was isolated and sequenced, revealing a Ixt,dicled wimm~ el 519 amino acids (61.974 kDa) with an additional .signal peptide oi 20 amino acids. 80% el amino acids from residms 1-474 were identical when compared with the human lAP, but there was ~ 31% idcnti~ in the COOg-i-4~mlnal 45 amino acids. The homo~gy diverges just belore the putative binding site for the phosphatid~linositoi-gl~ca~ (PI-gl~am) anchor. The resulting peptide in rat AP cootaias five hy(bophili¢ amino midis not larese~ in the imimmy slmt'ttm~ el human lAP. Binding el a synthetic 4g.mer encoding • portion el this unique aml divergem rngim (reskk.s 476-491) was compared with that of the full-length clone on Norlhem ~ of ra[ i m ~ RNA. Two aaRNAs, 3.0 / 2.7 klk were detected by both prolxes, confirming earlier results, but the 4g.ater bound prd,,eminll~ to the 3.0 kb mRNA. Tim protein produe/el the f u l l - k ~ h eDNA in a eeB-free syslem was 62 ki)a, ~ with the smaller e l ~ two lAP proteins wodm-ed by rat duodenal RNA. The eDNA tnmsfecled into COS-I cells imadut'ed • lAP *dust was released by phesl~batidylinositol-sfecific phospholilmse (PI-PLC). These data provide definitive that lAP is am-lmr~ by Pl-glycan and conclusively demonstrate that the unique COOH-terminal structure encoded 19. this rat mRNA suptmrts the addition o[ a Pl-glycan anchor.
Introduction Intestinal alkaline phosphatase (lAP) in the rat is unique among mammalian IAPs in that much activity is released into the serum during fasting and following fat feeding [1,2]. Consistent with this enhanced secretion is the production of two lAP products from cell-free translation of rat intestinal mRNA, 62 and 65 kDa 131. and the presence of two mRNAs, 3.0 and 2.7 kb dete~ted by using a human PLAP probe [4]. Our i:revious
]'he ~quence daw in Ihi~ paper have hewn submittt.~d to the EMBL ' Genbank Data Libranc-s under the a c c e ~ o n number X 17611 Abbre,,latJons: {I) AP. (int~linal) alkaline phosphatase: PLAP. placemal alkahne phosphatase; L / B / K . li,,er, bone. k~dno (alkahne phosphalase): kb. kdobase(s): Pl-glycan. ?hosphatld,,hno>ttol-gl?,can: IL~,P. rat alkaline pho~phatas¢; PI-PLC. Pl-.,,pccifl~ phosphohpa~¢ (': VS(;. s'ariablc surface gl~ct~'>rotenn.
(',+rr~'%n'md.':.n.'.'-':: M I.O~,e. D~partment ~.~f Pe~hatncs. Wa.,,hmgton (.'anscrslt}. lk),t 8094. 600 S. Eu~:Ind Ave.. SI. Louis,. MO 63110. ( SA 0167-4838/~I
work suggested that the 62 kDa cell-free product might correspond with the tissue-'soluble" form (58-60 kDa) and the serum lAP, whereas the 65 kDa prt~luct was the membrane-bound tissue lAP. Rat lAP is released from membranes by PI-specific phospholipase C (PI-PLC) and is probably attached :o the membrane via a P! glycan anchor, as is the case for other alkaline pho:-.~hatases (AP) [5]. However. the amount of rat lAP released from the membrane by PI-PLC is not as great as for other APs, raising the possibility that rat IAP may be anchored in the m ~ n brane by other means [6]. Delineation of the primary structure of h u m a n APs has not allowed prediction of the attachment site for the Pl-glycan anchor, nor for the portion of the COOH-terminus of the protein necessary for such processing [5]. Asp-484 is the anchoring residue for human piacemat ~ r trLAP), and aspaJLic acid, serine and cysteine have been reported as anchoring sites in other proteins [7]. A sequence of hydrophobic residues in the COOH terminus of nascent PLAP seems required fol processing of the phosphatidylinositof glycan anchor [7]. The secreted rat IAP may be pro-
$03 50 ," 19
BBAPRO
B,,~ h~mi¢ a e~ B~,Jph~,,tt'u .4t ra. 1037 ( I qqO) 170-177 El,,¢v~o"
33534
Molecular c!oning and expression of a c D N A encoding the membrane-associated rat intestinal alkaline phosphatase M a r k L o w e ~, A r n o l d W. S t r a u s s -"~, R u t h A i p e r s ~ S h a k u n t l a S e e t h a r a m a n d D a v i d H. A l p e r s t Departotenls of t Medwme. : Ped~utr,'s
und
~ B~olog~¢al
(.'hem~tr)'. 14"ash~ngto~r L'nwer~t~ Med~(al ~ k o o ~ S t l~u~s. M O I U S A )
(Received ~ Jul} i98~))
Key words: Alkaline phosphala~: Phosphaudylnno~ttol
Rat intestinal alkaline phosphatase (lAP) has been pmilied and Woteolytic fragments sequenced. A eDNA h'larmT was constructed from duodenal poly(A)* RNA and screened for lAP positive clones by a f~l-length eDNA ¢kmc--eocot~l~ human lAP. A full length rat LAP ~ (2237 bp) was isolated and sequenced, revealing a Ixt,dicled wimm~ el 519 amino acids (61.974 kDa) with an additional .signal peptide oi 20 amino acids. 80% el amino acids from residms 1-474 were identical when compared with the human lAP, but there was ~ 31% idcnti~ in the COOg-i-4~mlnal 45 amino acids. The homo~gy diverges just belore the putative binding site for the phosphatid~linositoi-gl~ca~ (PI-gl~am) anchor. The resulting peptide in rat AP cootaias five hy(bophili¢ amino midis not larese~ in the imimmy slmt'ttm~ el human lAP. Binding el a synthetic 4g.mer encoding • portion el this unique aml divergem rngim (reskk.s 476-491) was compared with that of the full-length clone on Norlhem ~ of ra[ i m ~ RNA. Two aaRNAs, 3.0 / 2.7 klk were detected by both prolxes, confirming earlier results, but the 4g.ater bound prd,,eminll~ to the 3.0 kb mRNA. Tim protein produe/el the f u l l - k ~ h eDNA in a eeB-free syslem was 62 ki)a, ~ with the smaller e l ~ two lAP proteins wodm-ed by rat duodenal RNA. The eDNA tnmsfecled into COS-I cells imadut'ed • lAP *dust was released by phesl~batidylinositol-sfecific phospholilmse (PI-PLC). These data provide definitive that lAP is am-lmr~ by Pl-glycan and conclusively demonstrate that the unique COOH-terminal structure encoded 19. this rat mRNA suptmrts the addition o[ a Pl-glycan anchor.
Introduction Intestinal alkaline phosphatase (lAP) in the rat is unique among mammalian IAPs in that much activity is released into the serum during fasting and following fat feeding [1,2]. Consistent with this enhanced secretion is the production of two lAP products from cell-free translation of rat intestinal mRNA, 62 and 65 kDa 131. and the presence of two mRNAs, 3.0 and 2.7 kb dete~ted by using a human PLAP probe [4]. Our i:revious
]'he ~quence daw in Ihi~ paper have hewn submittt.~d to the EMBL ' Genbank Data Libranc-s under the a c c e ~ o n number X 17611 Abbre,,latJons: {I) AP. (int~linal) alkaline phosphatase: PLAP. placemal alkahne phosphatase; L / B / K . li,,er, bone. k~dno (alkahne phosphalase): kb. kdobase(s): Pl-glycan. ?hosphatld,,hno>ttol-gl?,can: IL~,P. rat alkaline pho~phatas¢; PI-PLC. Pl-.,,pccifl~ phosphohpa~¢ (': VS(;. s'ariablc surface gl~ct~'>rotenn.
(',+rr~'%n'md.':.n.'.'-':: M I.O~,e. D~partment ~.~f Pe~hatncs. Wa.,,hmgton (.'anscrslt}. lk),t 8094. 600 S. Eu~:Ind Ave.. SI. Louis,. MO 63110. ( SA 0167-4838/~I
work suggested that the 62 kDa cell-free product might correspond with the tissue-'soluble" form (58-60 kDa) and the serum lAP, whereas the 65 kDa prt~luct was the membrane-bound tissue lAP. Rat lAP is released from membranes by PI-specific phospholipase C (PI-PLC) and is probably attached :o the membrane via a P! glycan anchor, as is the case for other alkaline pho:-.~hatases (AP) [5]. However. the amount of rat lAP released from the membrane by PI-PLC is not as great as for other APs, raising the possibility that rat IAP may be anchored in the m ~ n brane by other means [6]. Delineation of the primary structure of h u m a n APs has not allowed prediction of the attachment site for the Pl-glycan anchor, nor for the portion of the COOH-terminus of the protein necessary for such processing [5]. Asp-484 is the anchoring residue for human piacemat ~ r trLAP), and aspaJLic acid, serine and cysteine have been reported as anchoring sites in other proteins [7]. A sequence of hydrophobic residues in the COOH terminus of nascent PLAP seems required fol processing of the phosphatidylinositof glycan anchor [7]. The secreted rat IAP may be pro-
$03 50 ," 19
