Many tissues such as muscle, skin, liver, and peripheral nerve, have remarkable ability to repair and regrow after injury. However, the CNS (brain and spinal cord) is limited in its ability to repair or regrowth causing permanent brain damage or paralyses. It has been suggested that neuronal regeneration is inhibited by some unidentified factors released from the adult non-neuronal glial cells (oligodenddrocytes and astrocytes). Two proteins of 35 and 250 kDa (designated as IN-1) were identified whose immunoinhibition allowed neuronal regeneration. Injections of IN-1 into adult rats after spinal-cord injury also allowed some neuronal growth across the injured tissue. However, the identity of the proteins targeted by IN-1 remained obscured.

Most recently an inhibitory myelin protein, Nogo (Neurite outgrowth), has been cloned and characterized from human and rat. Nogo is a potent inhibitor of neurite outgrowth and may help block the regeneration of the CNS. Nogo is the 4th member of reticulon (Rtn) family, which contains a transmembrane domain of unknown function. There are three alternative isoforms of Nogo, designated Nogo-A (full length human protein 1192 aa; calculated mol wt 135 kDa; rat 1163 aa), an intermediate form Nogo-B (373 aa; ~37 K, lacks 186-1004 aa within the extracellular domain), and a shorter form Nogo-C (199 aa; ~25 K, similar to rat vp20 and foocen-s; lacks 186-1004 aa but which has a smaller, alternative N-terminal domain). Nogo-A has a putative extracellular domain of 1024 AA, 2-3 TM domains, and a short C-terminus of 43 AA. Nogo-A is localized to the CNS-myelin, and is highly expressed in oligodendorcytes but not by Schwann cells. Nogo-B and Nogo-C have been found in several non-neuronal tissues (skeletal muscle, kidney, skin, lung, and spleen), and one of them may be the 35-kDa protein recognized by IN-1 antibody.

Full length Nogo-A has the strongest inhibitory activity and it may be the 250-kDa protein recognized by the IN-1 antibody. The N-terminus of Nogo A is unique, whereas the C-terminus has sequence homology with the reticulon family. Nogo-A has endoplasmic reticulum retention signal sequence. It is not clear how Nogo-A contacts axons, and reaches the membrane of oligodendorcytes. A 66-aa hydrophilic region of Nogo, located between the two TM domains, has the most inhibitory properties of Nogo. In contrast to Nogo, Rtn 1, -2, and 3 do not inhibit axonal regeneration. A 25-aa inhibitory peptide sequence (designated as Nogo-P4; rat Nogo 1056-1080 aa) or 31-55 AA of the 66 AA active peptide) appears to sufficient to produce core inhibitory properties of Nogo. This 66-aa region also has the least similarity to Rtn proteins. The corresponding Rtn-P4 peptide sequence has no activity.

Recently, a brain specific leucine-rich-repeat protein with high affinity for soluble Nogo-66, termed Nogo receptor (Ngr) has been cloned and characterized. Cleavage of Ngr from the axonal surface renders neurons insensitive to Nogo-66. Ngr expression is sufficient to impart Nogo-66 axonal inhibition to unresponsive neurons. Ngr protein (mouse, rat, monkey, and human 473 aa; chromosome 22q11) contains a signal sequence followed by eight LRR domains, an LRR cysteine-rich CT-flanking domain, and unique GPI anchorage site. Human and mouse Ngr proteins are ~89% identical. Ngr is expressed in brain and lower levels are also detected in hear and kidney but not in other peripheral tissues. Ngr has been localized to axons. A peptide sequences 1-40 aa of the Nogo-66, designated NEP1-40, acts as competitive antagonist of NgR and blocks Nogo-66 or CNS myelin inhibition of axonal outgrowth in vitro. Intrathecal administration of NEP1-40 to rats with mid thoracic spinal cord hemisection results in significant axon growth of corticospinal tract.

Additional proteins homologous to Ngr have now been identified and designated NGR2 or NgRH1 (nogo-66 receptor homolog-1; human and rat 420 aa; chromosome 11q12.1 ) and Ngr3 or NgRH2 (nogo-66 receptor homolog-2; rat 438 aa; mouse, 445 aa; human 441 aa, chromosome 17p13.3). There is approx. 45% sequence homology between the NGR1-3. Despite high degree of sequence homology and similar protein topology, the NGR2 and NGR3 do not bind Nogo, MAG, Omgp or NGR1. The identity of other natural ligands for NGR2-3, and the physiological roles of NGR2-3 are unknown. The NGR2-3, like NGR1, display 8-LRRs flanked by LLRCT and LRRNT, and a C-terminal GPI signal sequence. The LRR 1-2 regions are 24-aa long and the LRR-3 region is 25-aa long. Both NGR2 and NGR3 are expressed in the brain. Significant level of NGR2 expression was detected in liver. Other peripheral tissues also show some level of NGR2. The NGR2-3 proteins appeared as ~45 and ~65 kda protein (glycosylated).

ADI has produced highly specific rabbit antibodies for rat Nogo-A, Nogo-B, and Nogo-C. These antibodies do not crossreact with each other and can be used to study various Nogo proteins. Respective antigenic or control peptides are also available to confirm specificity of antibodies. In addition, synthetic inhibitory peptides Nogo (Nogo-P4) and the corresponding control peptide from Rtn (Rtn-P4) to further explore the biological effects of these active peptides.

 

m=mouse; r=rat; h=human; c=chicken; f=frog; ~CT or ~NT=near C or N-terminus.

* Expected antibody crossreactivity information is mostly based upon high (>70%) sequence conservation of antigenic/control peptides in various species. When antibody crossreactivity has actually been experimentally confirmed in various species, it will be mentioned in the appropriate data sheets.

"Neat Antisera or antisera" are the unpurified antiserum and it is suitable for ELISA and Western.
"Affinity pure" IgG may be more suitable for immunohistochemical (IHC) applications and to reduce background in most immunological applications including ELISA and Western.
"Control peptides" can not be used for Western as they are very short peptides. They are intended for ELISA or antibody blocking studies to establish antibody specificity.
Western blot +ve protein controls, where available, are semi-pure, pure or recombinant proteins that are formulated in SDS-PAGE sample buffer (reduced). They are recommended to be used for Western (load 10 ul/lane) for visulization with antibodies.
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List of Publications using ADI's antibodies for Nogo's.

Nogo-P4 Yamashita T 2003 Nature Neuroscience 6, 461 - 467 The p75 receptor acts as a displacement factor that releases Rho from Rho-GDI neurite outgrowth assay.

Nogo-A Tozaki H 2002 Mol. Brain Res. 104, 111-119 Expression of Nogo protein by growing axons in the developing nervous system IHC rat brain, identical staining with mAB.

Nogo-A Wang KC 2002 Nature 417, 941-944 Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth WB, OMG from R7D.

Notes: Antibodies usage is indicated in the following techniques: WB=Western Blot ; IHC-Immunohistochmistry; IP=Immunoprecipition; Flow=Flow cytometry;