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Complete Amino Acid Sequence of Human Plasma Zn-A2-Glycoprotein and Its Homology to Histocompatibility Antigens

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Complete Amino Acid Sequence of Human Plasma Zn-A2-Glycoprotein and Its Homology to Histocompatibility Antigens Proc. Nati. Acad. Sci. USA Vol. 85, pp. 679-683, February 1988 Biochemistry Complete amino acid sequence of human plasma Zn-a2-glycoprotein and its homology to histocompatibility antigens (plasma protein structure/glycan structure//HLA dass I antigens/secretory major histocompatibility complex-related protein/immnnoglobuiln gene superfamily) TOMOHIRO ARAKI*, FUMITAKE GEJYO*t, KEIICHI TAKAGAKI*, HEINZ HAUPT*, H. GERHARD SCHWICKt, WILLY BURGI§, THOMAS MARTI¶, JOHANN SCHALLER¶, EGON RICKLI¶, REINHARD BROSSMER11, PAUL H. ATKINSON**, FRANK W. PUTNAMtt, AND KARL SCHMID*# *Department of Biochemistry, Boston University School of Medicine, Boston University Medical Center, Boston, MA 02118; *Behringwerke AG, 3550 Marburg/Lahn, Federal Republic of Germany; lZentrallaboratorium, Kantonsspital, 5001 Aarau, and lInstitut fOr Biochemie, University of Berne, 3012 Berne, Switzerland; IlDepartment of Biochemistry, University of Heidelberg Medical School, 6900 Heidelberg, Federal Republic of Germany; **Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, NY 10461; and ttDepartment of Biology, Indiana University, Bloomington, IN 47405 Contributed by Frank W. Putnam, September 22, 1987 ABSTRACT In the present study the complete amino acid like determinant in common with nephritogenic urinary sequence of human plasma Zn-a2-glycoprotein was deter- glycoproteins (2, 4). mined. This protein whose biological function is unknown In this paper we describe the determination of the com- consists of a single polypeptide chain of 276 amino acid plete amino acid sequence of Zna2gp, including the location residues including 8 tryptophan residues and has a pyroglu- of the two disulfide bridges and the structure of the three tamyl residue at the amino terminus. The location of the two carbohydrate units, and we discuss the possible secondary disuhlde bonds in the polypeptide chain was also established. structure of this protein. An unexpected finding made by The three glycans, whose structure was elucidated with the aid computer analysis of the sequence data is that Zna2gp is of 500 MHz 1H NMR spectroscopy, were sialylated N- closely related to antigens of the major histocompatibility biantennas. The molecular weight calculated from the poly- complex (MHC) in amino acid sequence and in domain peptide and carbohydrate structure is 38,478, which is close to structure. This suggests that Zna2gp may have a role in the the reported value of -41,000 based on physicochemical expression of the immune response. measurements. The predicted secondary structure appeared to be comprised of 23% a-helix, 27% 13-sheet, and 22% 13-turns. MATERIALS AND METHODS The three N-glycans were found to be located in 1-turn re- gions. An unexpected finding was made by computer analysis Materials. The details of the preparation of the immuno- of the sequence data; this revealed that Zn-a2-glycoprotein is chemically homogeneous Zna2gp procedure will be pub- closely related to antigens of the major histocompatibility lished elsewhere. In brief, pooled normal human plasma was complex in amino acid sequence and in domain structure. fractionated with the Cohn ethanol procedure (1); the result- There was an unusually high degree of sequence homology ing supernatant of Cohn fraction V was concentrated by with the a chains of class I histocompatibility antigens. ultrafiltration followed by ammonium sulfate fractionation, Moreover, this plasma protein was shown to be a member of zone electrophoresis, and gel filtration. The remaining traces the immunoglobulin gene superfamily. Zn-a2-glycoprotein of serum proteins were successfully removed using solid- appears to be a truncated secretory major histocompatibility phase immunoadsorption employing antisera against human complex-related molecule, and it may have a role in the serum and human a,-acid glycoprotein. A yield of 400 mg of expression of the immune response. homogeneous Zna2gp was obtained from 100 liters of plasma. The preparation used was number 050584 from Zn-a2-glycoprotein (Zna2gp) (Mr =38,500) is one of several Behringwerke. human plasma proteins of unknown function that have been Methods. Prior to cleavage, Zna2gp was enzymatipally highly purified and characterized in terms of chemical and desialized using Vibrio cholera neuraminidase (Calbiochem) physical chemical properties (1, 2). This protein was first followed by reduction and alkylation with iodoacetic acid. isolated from fraction VI ofthe Cohn ethanol procedure, and Specific chemical cleavage was done initially with CNBr at its name derives from the fact that it was precipitated by 40C in 70% (vol/vol) formic acid, followed by a second addition of zinc ions (1). The purified protein appeared cleavage in 70%o (vol/vol) formic acid at room temperature to homogeneous on electrophoresis and on amino-terminal split an Asp-Pro bond, and then by incubation in dilute HCl amino acid analysis, which revealed a blocked end group. (0.012 M, 23TC) to break an Asp-Thr bond. Enzymatic However, polymorphism of Zna2gp was observed electro- digestion was carried out with the following enzymes: tryp- phoretically both for the native and the asialo form of the sin, lysyl-endopeptidase, Staphylococcus aureus V8 prote- protein (3). The carbohydrate content was reported to be ase, chymotrypsin, proline-specific endopeptidase, carboxy- 18% and to consist of N-acetylneuraminic acid, galactose, peptidase Y, and pyroglutamate aminopeptidase. The puri- mannose, fucose, and N-acetylglucosamine (1). The lack of fication of the resulting peptides was carried out by N-acetylgalactosamine indicated the absence of O-glycans. reversed-phase HPLC (Varian model 5000 liquid chromato- Although the possible biological function of this protein is graph with an Altex-Ultrasphere-ODS or Toyo-Soda TSK- not known, it has been speculated that Zna2gp has a lectin- Abbreviations: Zna2gp, Zn-a2-glycoprotein; MHC, major histocom- patibility complex. The publication costs of this article were defrayed in part by page charge TPresent address: Second Department of Internal Medicine, Niigata payment. This article must therefore be hereby marked "advertisement" University School of Medicine, Niigata, Japan. in accordance with 18 U.S.C. §1734 solely to indicate this fact. #*To whom reprint requests should be addressed. 679 680 Biochemistry: Araki et al. Proc. Natl. Acad. Sci. USA 85 (1988) 10 20 30 <Gln-Glu-Asn-GlnAs-Gly-r r-T-7yr-Ser-Leu-7tw-Tyr-Ile- Tyr-7hr-Gly-Lau-Sr-Lys-His-Val-Glu-Asp -Val-Pro Ala-Phe-Gln-Ala-Leu- _V - - - __I - -- -- I - I.- II-. -- TI-I TI-2 Vi 1 - V2 I - CBUC1 - I-CBIICZ 1 I CBIIC3 CTI 40 S0 60 Gly-Ser-Leu-Asn-Asp-Leu-Gln-O-Phe-PhArg-T iyr-ASn-S r-LYS-A -AArg-LYSSr-Gln-Pr-t-Gly-LeuTp-ArgqGln-Val-Glu-Gly-Met- I CBIV - I - TIl-3 - 1 l - I ~~~CB]ICS I LE1 1ILE2 I - CBIC4 - 1 I CBIC6 - 11 - CT2 - 70 so TI-4TI~~~~BI4---- --4---- - IT I LE3 1ILE4 I I - V3 - - V4 - 1 100 _ 110 120 Gly-Sr-His-Vallu-Gln-Gly-Arg-Ru-yO-l-lyelo l-Gly-Slr-TyrlaPtyTp-yTyr-Tyr-Tyr-AmpGly-Lys- 11 I - TI-6 - - TI-7 - LES - - LE6 - VS --- I V6 130 140 150 Anp-yr-IleGlu-PIAsn-Lys-Glu-le_.PrAla-TzpVal-PPoN-mpPoAla-Ala-Gln-Imrlys>Glnt-Ly Lys-TrpGlu-Ala-lu-Prc> TI-8 - I9TI-9l LE7 -E$ILE8 -_ __ _ __ _ _ II V7 160 I 170 180 Val-br5aTqAyLyAa>-_PoAl-~-~qL>trVal u-yoiyr-yla-u-Asp -4 I - TI-li I I-TI - 12-4 TI - 13-4 LE9 - LflO I I LEl_ -LEl_ I - V8 190 200 I 210 Arg-Gln-AspPro-Pro-Ser-Va]L-Val-Val-TwShrr41sGln-Ala-Pro-Gly-Glu-Lss.-ys-Loyu-Lys-Cysu-LyuAla-Tr-AspPho-Tyr-Pro- 7~~~~~~I -- -- -- 7F 7l 7I ,- 7- 777777 I ~~~~~~TII-l TIE-2 LE13 ILE14 V9 220 230 240 Gly-Lys-Ile-Asp-Val-His-Tmprt-Arg Ala-Gly-Gln-Val-Gln-Glu-Pro Glu-Lo u-Arg-Gly-AspVal-Ieu-HAis-AGly-AnGly-lthr-Tiyr- -7-7,7 -7 - 7 --'-7-7-7-7 -7 7 -7---7-7 -7 -7777 -I! - TB -3 - 1 TB-4 - -4I I - ~~~~~~~~LElLE1S.----------- ----------- I - TB SC1 - 1 250 260 270 Gln-Ser-rpVal-Val-Val-Ala-Val-Pro-Pro Gln-Asp-Tr-Ala-Pro-Tyr-Ser-Cy-is-Val-Gln-His-Swr-Swr-Ieu-Ala-Gln-Protou-Val- Vii F-TBSC2---i --- TSC3 -- TISC4 - - 276 MAC-I Val-Pro-TrpGlu-Ala-Ser-COXH -T SCS-4 I- P1 FIG. 1. The complete amino acid sequence of human plasma Zna2gp. CB, cyanogen bromide cleavage fragments; TI, tryptic peptides of acid-cleavage fragment I; TII, tryptic peptides of acid-cleavage fragment II; LE, lysyl-endopeptidase peptides; V, S. aureus V8 protease peptides; CT, tryptic peptides ofcitraconylated CBII; MAC, mild-acid-cleaved peptides; P1, proline-specific endopeptidase peptide of MAC-1; -, carboxypeptidase Y; and -, pyroglutamate aminopeptidase digestions ofZna2gp. Solid lines indicate those regions ofthe peptides that were sequenced, whereas the dotted lines indicate residues or regions ofpeptides that were not sequenced. Disulfide bonds are indicated by the bold lines with -S-S-. The solid squares above Asn-90, Asn-106, and Asn-237 indicate N-glycans. In this figure the amino acid sequences of only the major peptides are included, but it should be noted that additional sequences were established to confirm the position of each amino acid residue. 120 C18 column and a Bio-Rad Hi-Pore C4 column). Auto- resulting glycopeptides were purified to homogeneity by mated sequence determination was performed with a Beck- HPLC. They were characterized by their amino acid se- man 890C sequencer, while for the manual sequence deter- quences and by determining the structure of the carbohy- mination the dimethylaminoazobenzene isothiocyanate (DA- drate moiety employing 500 MHz 'H-NMR spectroscopy by BITC) procedure was used (5). methods described (7) except that a Varian VXR 500 MHz For the location of the disulfide bonds, asialo Zna2gp was spectrometer was used with 32K data blocks.
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