The endless story of the glial fibrillary acidic protein

1994 ◽  
Vol 107 (8) ◽  
pp. 2299-2311 ◽  
Author(s):  
W.J. Chen ◽  
R.K. Liem
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Intermediate Filament ◽  
Self Assembly ◽  
Acidic Protein ◽  
Terminal Deletion ◽  
Deletion Mutants ◽  
Tail Domain ◽  
Intermediate Filament Proteins ◽  
Type Iii ◽  
Head Domain

All intermediate filament proteins consist of an alpha-helical rod domain flanked by non-helical N-terminal head and C-terminal tail domains. The roles of the non-helical domains of various intermediate filament proteins in the assembly and co-assembly of higher-order filamentous structures have been studied by many groups but with quite contradictory results. Type III intermediate filaments are unique in that they can form homopolymers both in vitro and in vivo. The expression and assembly characteristics of carboxy- and amino-terminal deletion mutants of glial fibrillary acidic protein (GFAP), an astrocyte-specific type III intermediate filament protein, were examined by transient transfections of either vimentin-positive or vimentin-negative variants of human adrenocarcinoma-derived SW13 cell lines. Whereas complete deletion of the C-terminal tail domain of GFAP results in the formation of polymorphic aggregates, both intranuclear and cytoplasmic in self-assembly experiments, efficient co-assembly of these tail-less GFAP mutants with vimentin can be achieved as long as the KLLEGEE sequence at the C-terminal end of the rod domain is preserved. Up to one-fifth of the C-terminal end of the tail domain can be deleted without affecting the capability of GFAP to self-assemble. The highly conserved RDG-containing motif in the tail domain may be important for self-assembly but is not sufficient. The entire head domain seems to be required for self-assembly. All N-terminal deletion mutants of GFAP share the same phenotype of diffuse cytoplasmic staining when expressed in vimentin-negative SW13 cells. Although co-assembly with vimentin can still be achieved with completely head-less GFAP, preservation of some of the head domain greatly enhanced the efficiency. Our results form the basis for further, more detailed mapping of the essential regions in filament assembly of GFAP and other type III IFs.

scholarly journals Roles of head and tail domains in alpha-internexin's self-assembly and coassembly with the neurofilament triplet proteins

1998 ◽  
Vol 111 (3) ◽  
pp. 321-333 ◽  
Author(s):  
G.Y. Ching ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Self Assembly ◽  
Deletion Mutant ◽  
Deletion Mutants ◽  
Intermediate Filament Protein ◽  
Intermediate Filament Proteins ◽  
Amino Terminal ◽  
Head Domain ◽  
Normal Filament ◽  
Filament Network

The roles of the head and tail domains of alpha-internexin, a type IV neuronal intermediate filament protein, in its self-assembly and coassemblies with neurofilament triplet proteins, were examined by transient transfections with deletion mutants in a non-neuronal cell line lacking an endogenous cytoplasmic intermediate filament network. The results from the self-assembly studies showed that the head domain was essential for alpha-internexin's ability to self-assemble into a filament network and the tail domain was important for establishing a proper filament network. The data from the coassembly studies demonstrated that alpha-internexin interacted differentially with the neurofilament triplet protein subunits. Wild-type NF-L or NF-M, but not NF-H, was able to complement and form a normal filament network with the tailless alpha-internexin mutant, the alpha-internexin head-deletion mutant, or the alpha-internexin mutant missing the entire tail and some amino-terminal portion of the head domain. In contrast, neither the tailless NF-L mutant nor the NF-L head-deletion mutant was able to form a normal filament network with any of these alpha-internexin deletion mutants. However, coassembly of the tailless NF-M mutant with the alpha-internexin head-deletion mutant and coassembly of the NF-M head-deletion mutant with the tailless alpha-internexin mutant resulted in the formation of a normal filament network. Thus, the coassembly between alpha-internexin and NF-M exhibits some unique characteristics previously not observed with other intermediate filament proteins: only one intact tail and one intact head are required for the formation of a normal filament network, and they can be present within the same partner or separately in two partners.

Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins

1999 ◽  
Vol 112 (13) ◽  
pp. 2233-2240
Author(s):  
G.Y. Ching ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Self Assembly ◽  
Chimeric Proteins ◽  
Filament Formation ◽  
Intermediate Filament Proteins ◽  
L Protein ◽  
Type Iv ◽  
Filament Assembly ◽  
Head Domain ◽  
Neuronal Intermediate Filament Proteins

Type IV neuronal intermediate filament proteins consist of alpha-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat alpha-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lalphaalpha and Malphaalpha, the chimeric proteins generated by replacing the head domain of alpha-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, alphaLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of alpha-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the alpha-internexin head domain is essential for alpha-internexin's ability to self-assemble. While coassembly of Lalphaalpha with NF-M and coassembly of Malphaalpha with NF-L resulted in formation of filaments, coassembly of Lalphaalpha with NF-L and coassembly of Malphaalpha with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.

scholarly journals Common and variant properties of intermediate filament proteins from lower chordates and vertebrates; two proteins from the tunicate Styela and the identification of a type III homologue

1998 ◽  
Vol 111 (19) ◽  
pp. 2967-2975
Author(s):  
D. Riemer ◽  
K. Weber
Keyword(s):  
Intermediate Filament ◽  
Self Assembly ◽  
Expression Patterns ◽  
Intron Position ◽  
Gene Organization ◽  
The Body ◽  
Specific Expression ◽  
Intermediate Filament Proteins ◽  
Type Iii

The chordates combine the vertebrates and the invertebrate phyla of the cephalo- and urochordates (tunicates). Two cytoplasmic intermediate filament (IF) proteins of the urochordate Styela plicata are characterized by cDNA cloning, gene organization, tissue specific expression patterns in the adult animal and the self assembly properties of the recombinant proteins. In line with metazoan phylogeny St-A and St-B have the short length version of the coil 1b domain found in all vertebrate and cephalochordate IF proteins while protostomic IF proteins have the longer length version with an extra 42 residues. St-A is the first IF protein from a lower chordate which can be unambiguously related to a particular vertebrate IF subfamily. St-A shares 46% sequence identity with desmin, displays the N-terminal motif necessary for filament assembly of type III proteins and forms normal homopolymeric 10 nm filaments in vitro. St-A but not St-B is present in smooth muscle cells of the body wall musculature. St-A and St-B are found as separate networks in some interior epithelia. St-B shares 30 to 35% identity with keratin 8, St-A and desmin and does not form IF under in vitro assembly conditions. Its relation to a particular vertebrate IF type or to the eight currently known IF proteins from the cephalochordate Branchiostoma remains unresolved. The striking relation between St-A and desmin predicts that the common progenitor of the urochordate (tunicate) and the cephalochordate/vertebrate lineages already possessed a type III homologue. Unlike in vertebrates intron patterns cannot be used to classify the tunicate IF genes. Although St-A is a type III homologue its gene shows an intron position which in vertebrates is restricted to keratin type II genes.

Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation

1989 ◽  
Vol 93 (1) ◽  
pp. 71-83
Author(s):  
R.A. Quinlan ◽  
R.D. Moir ◽  
M. Stewart
Keyword(s):  
Escherichia Coli ◽  
Glial Fibrillary Acidic Protein ◽  
Fusion Protein ◽  
Intermediate Filament ◽  
Coiled Coil ◽  
Acidic Protein ◽  
Vector System ◽  
Cdna Fragment ◽  
Intermediate Filament Proteins ◽  
Helical Domain

We have expressed in Escherichia coli a 1258 bp cDNA fragment corresponding to 97% of mouse glial fibrillary acidic protein (GFAP), the principal intermediate filament protein of astrocytes. High levels of expression were obtained, as a fusion protein with 32 residues of the bacteriophage lambda cII protein, using the pLcII expression vector system of K. Nagai and H.-C. Thogersen. Although removal of the cII protein fragment by proteolysis using factor X proved difficult, a protein corresponding to most of the cDNA fragment was obtained by cleaving at the endogenous thrombin site near the middle of the N-terminal non-helical domain of GFAP. A shorter 1047 bp fragment, in which the C-terminal non-helical domain of GFAP was deleted, was also produced using oligonucleotide-directed site-specific mutagenesis of the original cDNA clone. After proteolysis with thrombin, this material gave a fragment that corresponded to the alpha-helical coiled-coil rod region of the GFAP molecule, together with a portion of the non-helical N-terminal domain. The fragments produced were characterized both biochemically and ultrastructurally, and appeared to retain the conformation of native GFAP. Crosslinking showed that all fragments formed molecules containing two chains (‘dimers’) that associated to form four-chain molecular dimers (‘tetramers’) analogous to those formed by intact intermediate filament proteins. Shadowed preparations showed the presence of rod-like particles that closely resembled those observed for other intermediate filament proteins and proteolytically prepared rod domains. Remarkably, the fusion protein produced from the entire 1258 bp cDNA fragment and the cII peptide was able to form filaments that closely resembled those produced by native GFAP. However, fragments in which either the cII peptide or the C-terminal non-helical domain were removed, or in which both were removed, failed to form filaments under standard assembly conditions. Although preliminary in nature, these results suggest that both N- and C-terminal non-helical domains may have a role in intermediate filament formation. Moreover, the fragment corresponding approximately to the GFAP rod formed paracrystals similar to those observed with other coiled-coil proteins. The molecules in these paracrystals were arranged antiparallel with the two molecules in the unit cell, which may correspond to the four-chain molecular dimer (tetramer), overlapping by approximately two-thirds of their length.

Retinal functional alterations in mice lacking intermediate filament proteins glial fibrillary acidic protein and vimentin

2015 ◽  
Vol 29 (12) ◽  
pp. 4815-4828 ◽  
Author(s):  
Kirsten A. Wunderlich ◽  
Naoyuki Tanimoto ◽  
Antje Grosche ◽  
Eberhart Zrenner ◽  
Milos Pekny ◽  
...  
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Intermediate Filament ◽  
Acidic Protein ◽  
Intermediate Filament Proteins
Sign in / Sign up

Export Citation Format

Share Document