Expression and divalent cation binding properties of the novel chemotactic inflammatory protein psoriasin.
Psoriasin is a novel chemotactic inflammatory protein that possesses weak similarity to the S100 family members of Ca(2+)-binding proteins, and that is highly up-regulated in hyperproliferative psoriatic keratinocytes. Here we have used the psoriasin cDNA to express recombinant human (rh) psoriasin in Escherichia coli as a fusion protein containing a hexa His tag and a factor Xa cleavage site in the NH2-terminus. The protein was purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose, digested with factor Xa, further purified by ion-exchange chromatography and characterized by two-dimensional (2-D) gel electrophoresis and NH2-terminal sequencing. The ability of rh psoriasin to bind Ca2+, Zn2+, and Mg2+ was determined by dialysis experiments. We found that rh psoriasin may bind at least seven molecules of Ca2+ in KCl and several molecules in NaCl, with an affinity for the first bound molecule of 1.3-1.6 x 10(4) M-1. This indicates that psoriasin may cooperatively bind several molecules of Ca2+ when present in the extracellular space, or putatively, if localized in subcellular compartments where the concentration of Ca2+ is relatively high. At least eight molecules of Zn2+ were bound in KCl and four in NaCl, with an affinity just below 1 x 10(4) M-1 for the first molecule. Thus psoriasin does not bind significant amounts of Zn2+ at physiological concentrations. Mg2+ and Ca2+ are bound anti-cooperatively and binding of each of the ions (Ca2+, Zn2+, or Mg2+), is accompanied by conformational changes that move tyrosine residues to more hydrophobic areas.
Vorum
H
H
Department of Medical Biochemistry, University of Aarhus, Denmark.
Madsen
P
P
Rasmussen
H H
HH
Etzerodt
M
M
Svendsen
I
I
Celis
J E
JE
Honoré
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D005110:Extracellular Space / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D011485:Protein Binding; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013347:Subcellular Fractions / Q000737:chemistry
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D000075023:S100 Calcium Binding Protein A7; D009418:S100 Proteins; C089242:S100A7 protein, human