THIOL ESTER ROLE IN CORRECT FOLDING AND CONFORMATION OF HUMAN ALPHA(2)-MACROGLOBULIN - PROPERTIES OF RECOMBINANT C949S VARIANT

被引:16
作者
GETTINS, PGW
BOEL, E
CREWS, BC
机构
[1] VANDERBILT UNIV,SCH MED,CTR MOLEC TOXICOL,NASHVILLE,TN 37232
[2] NOVO NORDISK AS,DEPT MOLEC BIOL PHARMACEUT BIOTECHNOL,DK-2880 BAGSVAERD,DENMARK
关键词
ALPHA(2)-MACROGLOBULIN; THIOL ESTER; SITE-DIRECTED MUTAGENESIS; BABY HAMSTER KIDNEY CELL; HUMAN;
D O I
10.1016/0014-5793(94)80430-3
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
To determine the role of the thiol ester in the folding of human alpha(2)-macroglobulin (alpha(2)M) in the active conformation, we have characterized a recombinant variant of alpha(2)M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester-forming cysteine. C949S cr,M behaves like methylamine-treated plasma alpha(2)M, with correctly formed inter-subunit disulfide bridges, non-covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric alpha(2)M does not require a pre-formed thiol ester. Active alpha(2)M may form in vivo by a two-step process involving initial folding to give a structure resembling that of C949S alpha(2)M followed by thiol ester formation and a conformational change that gives the native active state.
引用
收藏
页码:276 / 280
页数:5
相关论文
共 21 条
[11]  
ISAAC L, 1992, J BIOL CHEM, V267, P10062
[12]   CLEAVAGE OF STRUCTURAL PROTEINS DURING ASSEMBLY OF HEAD OF BACTERIOPHAGE-T4 [J].
LAEMMLI, UK .
NATURE, 1970, 227 (5259) :680-+
[13]  
LIND T, 1993, J BIOL CHEM, V268, P20705
[14]  
NAKAMAYE KL, 1986, NUCLEIC ACIDS RES, V14, P8679
[15]  
PANGBURN MK, 1992, J BIOL CHEM, V267, P8584
[16]   5'-3' EXONUCLEASES IN PHOSPHOROTHIOATE-BASED OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS [J].
SAYERS, JR ;
SCHMIDT, W ;
ECKSTEIN, F .
NUCLEIC ACIDS RESEARCH, 1988, 16 (03) :791-802
[17]   DOMAIN-STRUCTURE OF HUMAN ALPHA-2-MACROGLOBULIN - CHARACTERIZATION OF A RECEPTOR-BINDING DOMAIN OBTAINED BY DIGESTION WITH PAPAIN [J].
SOTTRUPJENSEN, L ;
GLIEMANN, J ;
VANLEUVEN, F .
FEBS LETTERS, 1986, 205 (01) :20-24
[18]   COMMON EVOLUTIONARY ORIGIN OF ALPHA-2-MACROGLOBULIN AND COMPLEMENT COMPONENT-C3 AND COMPONENT-C4 [J].
SOTTRUPJENSEN, L ;
STEPANIK, TM ;
KRISTENSEN, T ;
LONBLAD, PB ;
JONES, CM ;
WIERZBICKI, DM ;
MAGNUSSON, S ;
DOMDEY, H ;
WETSEL, RA ;
LUNDWALL, A ;
TACK, BF ;
FEY, GH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1985, 82 (01) :9-13
[19]   THE USE OF PHOSPHOROTHIOATE-MODIFIED DNA IN RESTRICTION ENZYME-REACTIONS TO PREPARE NICKED DNA [J].
TAYLOR, JW ;
SCHMIDT, W ;
COSSTICK, R ;
OKRUSZEK, A ;
ECKSTEIN, F .
NUCLEIC ACIDS RESEARCH, 1985, 13 (24) :8749-8764
[20]  
TAYLOR JW, 1985, NUCLEIC ACIDS RES, V13, P8765