Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: evidence that transporter undergoes endoproteolytic cleavage.
BACKGROUND
Serotonin transporter (SERT) is important target molecule for many antidepressive drugs and substances of abuse and is implicated in psychiatric disorders. We performed immunoblotting analysis of human and rat SERT in platelets and brain using the panel of eight site-specific SERT monoclonal and polyclonal antibodies (mAbs and pAbs).


METHODS
SDS-PAGE/Western blotting was conducted using peroxidase-labeled DEAE and affinity purified SERT antibodies under conditions preventing SERT post-extraction degradation.


RESULTS
Immunoreactive polypeptides of 14, 22, 32, 35, 37, 56, 68, and approximately 150-200 kDa were revealed in human platelet extracts using N-terminal and C-terminal SERT antibodies. In rat brain, C-terminal mAbs detected 68, 56, and 37 kDa proteins, in postmortem human brain predominated 35-37 kDa proteins. The immunoreactivity was abolished after antibody preadsorption with antigens. N-terminal pAbs recognized the 68 kDa protein, affinity purified on C-terminal mAbs, confirming its identity as full-size human SERT (the predicted size approximately 70.5 kDa).


CONCLUSIONS
The explanation of the results of immunoblotting most likely is a site-specific SERT endoproteolytic cleavage and a marked difference in glycosylation rather than nonspecific protein degradation, cross-reactivity with other epitopes or SERT alternative splicing.
Dmitriev
Alexander D
AD
Institute of Higher Nervous Activity and Neurophysiology of Russian Academy of Sciences, ul. Butlerova 5a, Moscow, 117485 Russia. dad75@rol.ru
Factor
Magnolia I
MI
Segal
Olga L
OL
Pavlova
Elena V
EV
Massino
Yulia S
YS
Smirnova
Maria B
MB
Yakovleva
Dinora A
DA
Dmitriev
Dmitriy A
DA
Kizim
Elena A
EA
Kolyaskina
Galina I
GI
Brusov
Oleg S
OS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001792:Blood Platelets / Q000737:chemistry*; D015153:Blotting, Western; D001923:Brain Chemistry; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D010447:Peptide Hydrolases / Q000502:physiology*; D011817:Rabbits; D051381:Rats; D050486:Serotonin Plasma Membrane Transport Proteins
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D010447:Peptide Hydrolases