Synthesis, isolation, and characterization of endogenous beta-galactoside-binding lectins in human leukocytes.
Galaptin, a beta-galactoside-binding lectin, was isolated from human buffy coat cells (peripheral leukocytes) and spleen by affinity chromatography. The molecular weight (32K) of the native buffy coat galaptin was similar to that for splenic galaptin. Their subunit molecular weight (14.5K), pI (4.60-4.85), and amino acid composition were identical. Both galaptins showed the presence of a single polypeptide when subjected to reversed-phase HPLC. Monospecific rabbit polyclonal antiserum raised against the 14.5-kDa subunit of splenic galaptin reacted with a 14.5-kDa polypeptide present in buffy coat cells, Epstein-Barr virus-immortalized B lymphoblastoid cells, and HL-60 promyelocytic leukemia cells. However, galaptin was not synthesized in vitro by buffy coat cells. Rather, a monomeric beta-galactoside-binding protein of Mr 15.5-16.5K that is immunologically distinct from galaptin was synthesized. This galactoside-binding protein was separable from galaptin by polyacrylamide gel electrophoresis and by anion-exchange chromatography. In contrast, immunoprecipitation experiments confirmed that galaptin was synthesized by the B lymphoblastoid cells. cDNA corresponding to the B lymphoblastoid cell mRNA encoding galaptin was amplified by the polymerase chain reaction. The amplified product was partially sequenced, and 299 nucleotides were identified. The derived amino acids corresponded to residues 6-65, 84-114, and 118-126 found to be present in human splenic galaptin. Immunohistochemical analyses revealed that galaptin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized to the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Allen
H J
HJ
Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, New York 14263.
Gottstine
S
S
Sharma
A
A
DiCioccio
R A
RA
Swank
R T
RT
Li
H
H
D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000737:chemistry; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002461:Cell Line, Transformed; D005697:Galactosides / Q000737:chemistry*; D037161:Galectins; D006388:Hemagglutinins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007962:Leukocytes / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011956:Receptors, Cell Surface; D013154:Spleen / Q000737:chemistry
D002352:Carrier Proteins; D005697:Galactosides; D037161:Galectins; D006388:Hemagglutinins; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins