A specific immunological probe for the major myelin proteolipid

Abstract

Major myelin proteolipid (MMPL, aiso called PLP) and DM-20 are the two major intrinsic membrane proteins of CNS myelin. A specific immunological probe was obtained for MMPL by raising antibodies against the synthetic tridecapeptide 117-129 of MMPL. Antibodies against this peptide reacted with the MMPL but dit not cross react with DM-20, while both proteolipids had been shown previously to be recognized by antibodies directed against the C-terminal hexapeptide of MMPL. This is in accordance with previous findings showing that DM-20 differs only from MMPL by a deletion of residues 100-140 (+ few units). Furthermore, this site-specific immunological probe also recognizes MMPL in its native form in oligodendrocytes in primary glial cell cultures.

The myelin proteolipid protein gene modulates apoptosis in neural and non-neural tissues

“…The question can be resolved by studying ) and PLP-deficient mice (ÀPLPtg). 16 Capase-3 þ cells are increased in jimpy and þ PLPtg mice but decreased in ÀPLPtg mice compared to control C57BL6 for jimpy, for þ PLPtg, and C57BL6 for ÀPLPtg mice. *Po0.05, **Po0.01, ***Po0.0001, compared to wild-type, unpaired t-test.…”

“…They were fixed with 4% paraformaldehyde in 0.1 M PBS at 41C for 30 min, rinsed twice in PBS/BSA for 5 min, treated with 0.25% Triton X-100 in PBS/BSA at room temperature (RT) for 30 min, rinsed in PBS/BSA for 5 min, then treated with 10% goat or 10% mouse serum in PBS/BSA at RT for 30 min, and rinsed in PBS/BSA for 5 min. They were incubated with rabbit polyclonal antibodies directed against the PLP-specific peptides 109-128, 16 as well as affinity purified rabbit polyclonal anti-PLP/DM20 directed against peptides 269-276 17 diluted 1 : 50 or 1 : 100. A mouse monoclonal anti-PLP/DM20 AA3 supernatant that recognizes the carboxy-terminus 18 was used 1 : 2 in a solution containing 0.1% Triton X-100 at 41C for 16-20 h on blastocysts, or blastocysts containing 0.1% Triton X-100 in PBS/BSA at 41C for 16-20 h were used as controls.…”

“…PLP and DM20 bands cut from the blastocysts and brain were sequenced and sequences match published databases for classical PLP. Western blots using an antibody that recognizes PLP but not DM20 16 detect PLP protein in the human trophoblast cell line, as well as the brain and myelin of wildtype mice but not in PLP-deficient transgenic mice (Figure 1c). Immunostaining of human trophoblasts in culture using a PLP-specific 16 or PLP/DM20 affinity purified poly-and monoclonal antibodies 17,18 shows weak staining in flattened cells but stronger staining in cells bearing migratory processes (unpublished data).…”

“…15 Total RNA loaded for blastocysts is approximately 1 3 that of brain. 51,52 (c) Homogenates prepared from the brain and myelin fractions of wildtype, PLP-deficient mice (ÀPLPtg), and human trophoblasts, cultured for 3 days probed with a polyclonal antibody that recognizes PLP but not DM20 isoform 16 show the appropriate band in the brain and myelin of wild-type mice and human trophoblasts 15 but not in ÀPLPtg mice separate and appropriate location of the message and DNA fragments in the cytoplasm and nucleus demonstrates that the DIG-labeled probes do not artifactually bind to the DNA internucleosomal fragments.…”

Aberrant splicing of proteolipid protein mRNA in the dysmyelinating jimpy mutant mouse.

“…Another proteolipid closely related to PLP is also found in reduced amounts in the myelin sheaths of oligodendrocytes. This proteolipid (DM-20) has identical amino acid sequences as PLP at the amino and carboxyl termini, but DM-20 is missing an internal domain of =40 amino acids (4)(5)(6).…”