Neuroserpin Portland (Ser52Arg) is trapped as an inactive intermediate that rapidly forms polymers

Abstract

The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by point mutations in the neuroserpin gene. We have shown a correlation between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relationship with the age of onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential interaction between the reactive centre loop of one neuroserpin molecule with b-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms loop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The Portland mutant has a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, with those of the CD analysis, are in keeping with the reactive centre loop of neuroserpin Portland being partially inserted into b-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclusions and the severity of dementia in FENIB associated with neuroserpin Portland. Moreover the inactivity of the mutant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in individuals with more severe forms of the disease.Keywords: conformational diseases; neuroserpin; polymerization; serpin; serpinopathies.The autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) results from point mutations in the neuroserpin gene and is characterized by inclusions of neuroserpin within cortical and subcortical neurons [1][2][3]. Neuroserpin is a member of the serine proteinase inhibitor or serpin superfamily. It inhibits the enzyme tissue plasminogen activator (tPA) and may be important in regulating neuronal plasticity and memory [4][5][6][7]. We have recently expressed, purified, and characterized wild-type neuroserpin and neuroserpin with the Ser49Pro mutation, which was identified in the first reported family with FENIB [6]. The mutation reduced the inhibitory activity of neuroserpin by 100-fold and increased the formation of polymeric protein under physiological conditions. Neuroserpin polymers result from the sequential insertion of the reactive centre loop of one molecule into b-sheet A of another [1,6]. The resulting species is inactive as a proteinase inhibitor and accumulates in the endoplasmic reticulum in cell models of disease [8] and in vivo [2].Three other mutants of neuroserpin are now recognized to cause FENIB: Ser52Arg, His338Arg and Gly392Glu [3]. This condition is unusual among neurodegenerative disorders in that there is a striking correlation between the number of inclusions within the cerebral cortex and an inverse relationship with the age of onset...

Update on alpha-1 antitrypsin deficiency: New therapies

“…Six mutations have now been described that underlie FENIB [48][49][50]. We have assessed a range of mutants of different severity in purified recombinant protein, cell, fly, worm and mouse models of disease [48,[51][52][53][54][55][56][57]. The data show a direct relationship between the severity of the mutation, the rate of polymer formation and the severity of the associated dementia.…”

Molecular mousetraps, α1-antitrypsin deficiency and the serpinopathies

“…This replacement of a consistently conserved residue in the shutter region resulted in large inclusions with affected family members dying by age 20 years. 83 The role of polymerisation in disease is supported in our demonstration that recombinant Ser49Pro neuroserpin has a greatly accelerated rate of polymerisation when compared to the wild type protein, [84][85][86] and that Ser52Arg, which causes a more severe clinical phenotype, polymerises even more rapidly. 85 The cellular handling of neuroserpin has been assessed by transiently transfecting COS cells with wildtype neuroserpin and mutants of neuroserpin that cause FENIB (Fig 5).…”

“…83 The role of polymerisation in disease is supported in our demonstration that recombinant Ser49Pro neuroserpin has a greatly accelerated rate of polymerisation when compared to the wild type protein, [84][85][86] and that Ser52Arg, which causes a more severe clinical phenotype, polymerises even more rapidly. 85 The cellular handling of neuroserpin has been assessed by transiently transfecting COS cells with wildtype neuroserpin and mutants of neuroserpin that cause FENIB (Fig 5). The most striking feature of the cell model is the retention of Syracuse (Ser49Pro) and Portland (Ser52Arg) neuroserpin as intracellular aggregates composed of polymers of mutant neuroserpin, similar to the loop-sheet polymers of mutant neuroserpin that can be isolated from the brains of individuals affected by FENIB.…”

pH‐dependent stability of neuroserpin is mediated by histidines 119 and 138; Implications for the control of β‐sheet A and polymerization

“…[17][18][19][20][21] Our previous work has shown that polymers of neuroserpin result from the sequential linkage between the reactive center loop of one molecule and b-sheet A of another. 7,22 However, it has recently been proposed that such polymers are formed by a b-hairpin linkage between not only the reactive center loop but also s5A inserting into b-sheet A of another molecule. 23 It is these polymers of neuroserpin that aggregate to form the cortical and subcortical periodic acid-Schiff-positive Collin's bodies that are the hallmark of the disease.…”

“…17,18,[24][25][26] There is a clear correlation between genotype and phenotype that can be explained on the rate of polymerization. 7,[18][19][20]22 Those mutants that polymerize most rapidly are associated with the greatest number of cerebral inclusions and the earliest age of onset of disease. Neuroserpin can also be inactivated by intramolecular loop insertion to form a latent conformer that has also been isolated from the brains of individuals with FENIB.…”