Design and structural characterisation of olfactomedin-1 variants as tools for functional studies

Abstract

BackgroundOlfactomedin-1 (Olfm1; also known as Noelin or Pancortin) is a highly-expressed secreted brain and retina protein and its four isoforms have different roles in nervous system development and function. Structural studies showed that the long Olfm1 isoform BMZ forms a disulfide-linked tetramer with a V-shaped architecture. The tips of the Olfm1 “V” each consist of two C-terminal β-propeller domains that enclose a calcium binding site. Functional characterisation of Olfm1 may be aided by new biochemical tools derived from these core structural elements.ResultsHere we present the production, purification and structural analysis of three novel monomeric, dimeric and tetrameric forms of mammalian Olfm1 for functional studies. We characterise these constructs structurally by high-resolution X-ray crystallography and small-angle X-ray scattering. The crystal structure of the Olfm1 β-propeller domain (to 1.25 Å) represents the highest-resolution structure of an olfactomedin family member to date, revealing features such as a hydrophilic tunnel containing water molecules running into the core of the domain where the calcium binding site resides. The shorter Olfactomedin-1 isoform BMY is a disulfide-linked tetramer with a shape similar to the corresponding region in the longer BMZ isoform.ConclusionsThese recombinantly-expressed protein tools should assist future studies, for example of biophysical, electrophysiological or morphological nature, to help elucidate the functions of Olfm1 in the mature mammalian brain. The control over the oligomeric state of Olfm1 provides a firm basis to better understand the role of Olfm1 in the (trans-synaptic) tethering or avidity-mediated clustering of synaptic receptors such as post-synaptic AMPA receptors and pre-synaptic amyloid precursor protein. In addition, the variation in domain composition of these protein tools provides a means to dissect the Olfm1 regions important for receptor binding.

Exposure to Tetrabutylammonium Bromide Impairs Cranial Neural Crest Specification, Neurogenic Program, and Brain Morphogenesis

“…The downregulation of olfm1 gene expression results in eye defects like reduction in size, retinal degeneration, defective optic nerve development, craniofacial defects, and abnormalities in the body axis. Thus, it plays a vital role in neural development and its derivatives. ,− Our results show that TBAB exposure results in the downregulation of the olfm1b Z isoform (red asterisk, Figure E,F). Hence, we infer that TBAB binding to AMPAR complex is the underlying basis of the reduction in olfm 1b Z isoform.…”

“…Olfactomedin is known to regulate the composition and function of the AMPAR complex. It interacts with the N-terminal domain of the AMPAR complex. ,, The zebrafish genome contains two olfactomedin genes, olfm1a and olfm1b . Olfm1a and 1b are preferentially expressed in the brain and retina during embryonic stages to adulthood.…”

Meta-Analysis of Transcriptomic Datasets Quantifies Differential Gene Expression, Affected Pathways, and Predicted Drugs from Hantavirus-Infected Human Material

“…As stated earlier, of the top 100 identi ed genes, only OLFM1, CCR7, KLRG1, and PTGDR2 have been identi ed in previous hantavirus publications. OLFM1, which produces olfactomedin and was found to be associated with hantavirus in a previous study, is also found to be highly expressed in neuronal tissue and may aid in nervous system development; however, relatively little is understood about many of its functions in other cell types [31]. Interestingly, while this gene is expressed in monocytes, its function is unknown and may warrant future investigation [32].…”

Regulatoryde novomutations underlying intellectual disability

“…Olfactomedin-1 ( OLFM1 ), is a secreted glycoprotein expressed at high levels in the brain (50). It plays role in neural progenitor maintenance, cell death in the brain, and the optic nerve arborization (35).…”