Phosphorylated human galectin-3: facile large-scale preparation of active lectin and detection of structural changes by CD spectroscopy.
Galectin-3 has a unique modular design. Its short N-terminal stretch can be phosphorylated, relevant for nuclear export and anti-anoikis/apoptosis activity. Enzymatic modification by casein kinase 1 at constant ATP concentration yielded mg quantities of mono- and diphosphorylated derivatives at Ser5/Ser11 in a 2:1 ratio. Their carbohydrate-inhibitable binding to asialofetuin, cell surfaces of three tumor lines, rabbit erythrocytes leading to haemagglutination and cytoplasmic sites in fixed tissue sections was not markedly altered relative to phosphate-free galectin-3. Spectroscopically, phosphorylation induced alterations in the far UV CD, indicative of an increase in ordered structure. This is accompanied by changes in the environment of aromatic amino acids signified by shifts in the near UV CD.
Kübler
Dieter
D
Biomolecular Interactions, German Cancer Research Center, Heidelberg, Germany. d.kuebler@dkfz.de
Hung
Chien-Wen
CW
Dam
Tarun K
TK
Kopitz
Jürgen
J
André
Sabine
S
Kaltner
Herbert
H
Lohr
Michaela
M
Manning
Joachim C
JC
He
Lizhong
L
Wang
Hui
H
Middelberg
Anton
A
Brewer
C Fred
CF
Reed
Jennifer
J
Lehmann
Wolf-Dieter
WD
Gabius
Hans-Joachim
HJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D047389:Casein Kinase I / Q000378:metabolism; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D060748:Fetuins; D005434:Flow Cytometry; D037502:Galectin 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007583:Jejunum / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007785:Lactose / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D012694:Serine / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D060748:Fetuins; D037502:Galectin 3; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012694:Serine; D000255:Adenosine Triphosphate; D047389:Casein Kinase I; D007785:Lactose