Methodological factors influencing measurement and processing of plasma reelin in humans

Abstract

BackgroundReelin, intensively studied as an extracellular protein that regulates brain development, is also expressed in a variety of tissues and a circulating pool of reelin exists in adult mammals. Here we describe the methodological and biological foundation for carrying out and interpreting clinical studies of plasma reelin.ResultsReelin in human plasma was sensitive to proteolysis, freeze-thawing and heating during long-term storage, sample preparation and electrophoresis. Reelin in plasma was a dimer under denaturing conditions. Boiling of samples resulted in laddering, suggesting that each of the 8 repeats expressed in reelin contains a heat-labile covalent bond susceptible to breakage. Urinary-type and tissue-type plasminogen activator converted reelin to a discrete 310 kDa fragment co-migrating with the major immunoreactive reelin fragment seen in plasma and also detected in brain. (In contrast, plasmin produced a spectrum of smaller unstable reelin fragments.) We examined archival plasma of 10 pairs of age-matched male individuals differing in repeat length of a CGG repeat polymorphism of the 5'-untranslated region of the reelin gene (both alleles < 11 repeats vs. one allele having >11 repeats). Reelin 310 kDa band content was lower in subjects having the long repeats in all 10 pairs, by 25% on average (p < 0.001). In contrast, no difference was noted for amyloid precursor protein.ConclusionsOur studies indicate the need for caution in measuring reelin in archival blood samples, and suggest that assays of plasma reelin should take into account three dimensions that might vary independently: a) the total amount of reelin protein; b) the relative amounts of reelin vs. its proteolytic processing products; and c) the aggregation state of the native protein. Reelin-plasminogen activator interactions may affect their roles in synaptic plasticity. Our results also suggest that the human CGG repeat polymorphism affects reelin gene expression, and may affect susceptibility to human disease.

Reelin expression and glycosylation patterns are altered in Alzheimer’s disease

“…Here we report consistent evidence not only of increased Reelin levels in CSF and affected brain areas of AD patients but also of increased transcriptional activity of the Reelin gene. Moreover, our data show that CSF storage and heating conditions are key factors in determining Reelin protein levels, in agreement with another study (24). Our covariance analyses further show that these differences are not attributable to age or gender (see Supporting Text and Table 1, which are published as supporting information on the PNAS web site).…”

“…7, which is published as supporting information on the PNAS web site). Similar to previous reports (13,24), the relative abundance of Reelin bands differed in plasma and CSF. Neither the intensity of the individual bands nor their relative banding pattern was altered in plasma from AD and mild cognitive impairment patients compared with NDC cases.…”

“…Reelin was determined essentially as described (24). Briefly, CSF (50 l), plasma (1.2 l), and brain (20 g) samples were analyzed on 6% SDS resolving gels.…”

Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region

“…It is known that reelin forms a covalent dimer via intermolecular disulfide bond(s) (16,22). In the present study, we determined the critical cysteine residue participating in the formation of the covalent dimer and showed that the dimer formation via this cysteine residue is required for the full biological activity of reelin.…”

“…Disulfide-linked oligomers have been detected in cell culture supernatant from 293T cells transiently transfected with full-length reelin, in the embryonic brain homogenates prepared from the cerebral cortex, and in human plasma (16,22). However, neither the number of reelin protomers contained in the functional oligomer nor the Cys residue(s) responsible for the cross-linking has been determined.…”