A Novel Brain-Specific Antigen: A Glycoprotein Electrophoretically Similar to but Immunochemically Different from Type B Nucleoside Diphosphatase1

Abstract

A monoclonal antibody, 1D4, recognizing a novel brain-specific protein was obtained. The 1D4 antigen is regarded to be a glycoprotein because it was adsorbed on the Con A-Sepharose column used for its purification. The antiserum (polyclonal antibodies) against the 1D4 antigen was raised in a rabbit and shown to react with just the same molecules as the 1D4 monoclonal antibody did. It was used to detect the antigen in crude tissue homogenates. The molecular mass of the 1D4 antigen was estimated to be 89 kDa by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the brain homogenate. The 1D4 antigen had multiple isoelectric points, the pattern of the bands detected on isoelectric focusing gel being quite similar to that of Type B nucleoside diphosphatase of the brain. However, they are distinct, since Type B nucleoside diphosphatase was not adsorbed by anti-1D4 antigen IgG-Sepharose 4B. The 1D4 antigen could not be detected in any of the peripheral organs or tissues tested. The 1D4 antigen was rich in the cerebrum, diencephalon, and cerebellum in the brain, and its content decreased with the distance of the region from the cerebrum. The amounts of the 1D4 antigen in the cerebrum and cerebellum increased with the respective developmental maturation. These findings suggest that the 1D4 antigen contributes to some brain-specific functions of the mature brain.

Activation of Protein-tyrosine Phosphatase SH-PTP2 by a Tyrosine-based Activation Motif of a Novel Brain Molecule

“…BIT, a novel antigen receptor-like molecule containing two TAMs is a major endogenous substrate of protein-tyrosine kinase(s) in crude brain suspensions 2 and is widely distributed in the brain in synapse-rich regions and some nerve fibers. 3 Predominant expression in the brain is a common feature of both BIT (9,10) and SH-PTP2 (34). Thus, the interaction reported here provides an important clue to the functions of BIT and SH-PTP2 in neural signal transduction.…”

“…Antibodies-The anti-BIT monoclonal antibody, named 1D4, and anti-BIT polyclonal antibodies which we used have been described (9,10). Polyclonal antibodies against SH-PTP2 and Grb2, and a monoclonal antibody against glutathione S-transferase (GST) were from Santa Cruz Biotechnology.…”

BIT, an immune antigen receptor‐like molecule in the brain1

“…The N‐terminal sequence of the purified BIT was also analyzed. Specific antibodies for BIT were isolated from rabbit anti‐BIT serum [2]using a column prepared with CNBr‐activated Sepharose 4B and purified BIT. The antibodies were used to screen a rat brain cDNA library in λgt11 (RL1043a, Clontech).…”

“…Rat tissues were homogenized in 20 mM Tris‐HCl (pH 6.8) containing 2% SDS and 2% 2‐mercaptoethanol, and the homogenates (10 μg protein) were resolved on a 10% SDS‐polyacrylamide gel, transferred onto a PVDF membrane (Millipore), and immunoblotted with anti‐BIT serum [2]. Detection proceeded using peroxidase–antiperoxidase complex as described previously [3]except that peroxidase activity was detected in the presence of 5 mg/ml of (NH 4 ) 2 Ni(SO 4 ) 2 ·6H 2 O for high sensitivity.…”

“…We previously found a brain‐specific glycoprotein in the rat brain using monoclonal antibody 1D4 [2, 3]. This glycoprotein postnatally increased and was highly expressed in the mature brain [2].…”