Properties and possible significance of substance P and insulin fibrils

Neuroscience Letters, 25 (1981) 321-325

321

Elsevier/North-Holland Scientific Publishers Ltd.

PROPERTIES AND POSSIBLE SIGNIFICANCE OF SUBSTANCE P AND INSULIN FIBRILS

ELAINE K. PERRY, ARTHUR E. OAKLEY* JOHN M. CANDY* and ROBERT H. PERRY* Department o f Neuropathology and *MRC Neuroendocrinology Unit, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE (U.K.)

(Received June 22nd, 1981; Accepted June 26th, 1981)

The neuropeptide substance P forms polymeric fibrils similar to those previously reported for the hormone, insulin. Structural and chemical aspects of these two fibrillary forms have been compared and their possible existence in vivo considered. Numerous substance P fibrils are readily formed in vitro in mM solutions under conditions which are physiological with respect to salt concentration, pH and temperature, whereas more severe conditions (heat and acid) are apparently required for the rapid formation of numerous insulin fibrils. Morphologically, both fibrils appear to be relatively long and unbranched and the neuropeptide fibrils are similar in size to such naturally occurring structures as neurofilaments. Disaggregation of the neuropeptide fibril follows dilution (1000-fold) whilst more stringent (alkaline) treatment is apparently necessary for insulin fibril dissociation. These observations are discussed in relation to the role of an insulin-like peptide in the formation of certain types of amyloid and the possibility that fibrillary or similar polymeric forms of substance P may exist in normal tissue. T h e reversible f o r m a t i o n o f fibrillary p o l y m e r s o f the p o l y p e p t i d e h o r m o n e insulin was first d e s c r i b e d o v e r 30 years a g o [9], a n d since then in v i t r o fibril f o r m a t i o n has been r e p o r t e d f o r several o t h e r h o r m o n e s , such as g l u c a g o n a n d calcitonin [3, 6]. F i b r i l l a r y f o r m s o f s m a l l e r n e u r o p e p t i d e molecules, such as the u n d e c a p e p t i d e s u b s t a n c e P, h a v e o n l y r e c e n t l y been identified [2] a n d in the present r e p o r t m o r p h o l o g i c a l a n d c h e m i c a l aspects o f s u b s t a n c e P a n d insulin fibrils are c o m p a r e d a n d their p o s s i b l e significance assessed. Insulin a n d s u b s t a n c e P were p u r c h a s e d f r o m S i g m a L o n d o n C h e m i c a l C o m p a n y L i m i t e d , U . K . T h e f o r m a t i o n o f insulin fibrils b y h e a t i n g a c i d i f i e d s o l u t i o n s a n d their d i s s o c i a t i o n a f t e r alkali t r e a t m e n t h a v e been p r e v i o u s l y described [9, 10]. These, a n d o t h e r t r e a t m e n t u s e d to test the f o r m a t i o n a n d d i s a g g r e g a t i o n o f s u b s t a n c e P a n d insulin fibrils, a r e detailed in T a b l e I. N e g a t i v e l y s t a i n e d specimens for e l e c t r o n m i c r o s c o p y were p r e p a r e d b y b l o t t i n g , air d r y i n g or c e n t r i f u g i n g ( 1 6 0 , 0 0 0 × g for 30 min) a 5 #l a l i q u o t o n t o f o r m v a r - c o a t e d nickel grids a n d then staining with 1 °70 p h o s p h o t u n g s t i c acid or u n b u f f e r e d 1 070 u r a n y l acetate. T o ascert a i n the c r o s s - s e c t i o n a l a p p e a r a n c e fibrils were fixed in 3070 g l u t a r a l d e h y d e , e m b e d d e d in e p o n , sectioned, m o u n t e d o n grids a n d s t a i n e d with s a t u r a t e d u r a n y l a c e t a t e in 5007o e t h a n o l a n d R e y n o l d s lead citrate. T h e grids were e x a m i n e d using a P h i l i p s 201 electron m i c r o s c o p e . 0304-3940/81/0000-0000/$ 02.50 © Elsevier/North-Holland Scientific Publishers Ltd.

322 TABLE I COMPARISON OF THE FORMATION AND DISPERSION OF SUBSTANCE P AND INSULIN FIBRILS Electron microscopic appearance Preparation Peptide a Solid, untreated Aqueous solution at 20 °C Saline (1-2°70 w/v) solution atl20°C Acid (50 mM HCL) saline solution at 20°C Acid saline solution at 85 °C, 15 min

Substance P

Insulin

Amorphous and Fibrillary aggregates Dispersed short fibrils (1 h)

Amorphous

Numerous long fibrils (1 h)

Occasional fibrils (2-3 days)

Numerous long fibrils (1 h)

Occasional fibrils (2-3 days)

Numerous long fibrils

Numerous fibrils

Fibrillary formes - treatmentb: Alkali (50 mM NaOH) 4°C, 18 h Numerous intact fibrils Dilution (1000-fold) with 1070 NaCI, 4°C, 48 h No fibrils seen Guanidine HCI (5 M), 20°C, 12 hVery few intact fibrils present Detergent - Non-ionic (1070 triton X-100) Numerous intact fibrils Anionic (2070 sodium laurylsulphate) No fibrils Acetonitrile (5007o w/v), 4 °C, 48 h No fibrils

Occasional fibrils (2-3 days)

No fibrils seen Intact fibrils Numerous intact fibrils Numerous intact fibrils

-

Numerous intact fibrils Numberous intact fibrils

aSolutions contained 5 mg/ml substance P or insulin, the latter dissolved in H20 or NaC1 after acidification and neutralization bSubstance P and insulin fibrils prepared from 5 mg/ml 207o saline solutions (plus acid and heat for insulin) and further treatments (except dilution) tested at this concentration.

T h e e f f e c t s o f v a r i o u s t r e a t m e n t s o n t h e f o r m a t i o n a n d d i s a g g r e g a t i o n o f substance P a n d insulin fibrils, assessed e l e c t r o n m i c r o s c o p i c a l l y , a r e s u m m a r i z e d in T a b l e I. S u b s t a n t i a l n u m b e r s o f s h o r t a g g r e g a t e d fibrils w e r e p r e s e n t in t h e c o m m e r cially a v a i l a b l e s o l i d f o r m a n d a l s o a q u e o u s s o l u t i o n s o f s u b s t a n c e P , b u t n o t t h o s e o f insulin. T h e r a p i d f o r m a t i o n o f n u m e r o u s l o n g fibrils o f s u b s t a n c e P in 1-2070 saline at r o o m t e m p e r a t u r e (Fig. 1A) did n o t o c c u r to a n y a p p r e c i a b l e e x t e n t w i t h insulin s o l u t i o n s w i t h o u t p r i o r h e a t a n d a c i d t r e a t m e n t (Fig. IB). F o r m a t i o n

of

s u b s t a n c e P fibrils in 1-2°70 saline was n o t g e n e r a l l y a c c o m p a n i e d b y a n y a l t e r a t i o n in the a p p e a r a n c e o f t h e s o l u t i o n , w h e r e a s f o r m a t i o n o f t h e i n s u l i n fibrils a f t e r h e a t and

acid

treatments

was,

as

previously

described

[9],

accompanied

by

the

323

B Fig. 1. Fibrillary forms of substance P (A) and insulin (B) stained with uranyl acetate. Magnifications are × 160,000 (A) and × 190,000 (B). The larger diameter insulin fibrils may consist of laterally aggregated smaller fibrils. (Figure reduced one-third in production.)

324

appearance of a flocculated, gel-like precipitate. Differences were also found in the relative stability of the respective fibrils (Table I). Insulin fibrils were apparently resistant to all of the treatments tested except 50 mM NaOH whereas substance P fibrils, which at high concentrations were stable to similar alkali treatment, disaggregated after dilution (1000-fold) or the addition of 2°7o sodium dodecylsulphate, 6 M guanidine or 50o7o acetonitrile (Table I). Comparing the outline of the two negatively stained fibrillary forms (Fig. 1A and IB) both are seen to be relatively long and unbranched but the outline of the neuropeptide fibril is consistently longer, straighter and more clearly defined. The insulin fibril, as previously reported [1], is apparently irregular, possibly due to twisting and varying degrees of lateral aggregation. In cross-section the substance P fibril (diameter 7 - 9 nm) had a distinctly stained outer edge surrounding a predominantly unstained interior, an appearance similar to that described for naturally occurring structures such as neurofilaments [8]. The cross-section of the insulin fibril was, in contrast, much more variable and less clearly defined and the diameter ranged from 3 to 15 nm. Preliminary X-ray diffraction studies of substance P fibrils have revealed a diffraction ring at 0.47 nm, which is similar to that reported for insulin [1] and suggests that a B pleated structure [7] is common to both fibrils. This common structural feature of the two fibrils may perhaps reflect an apparent resemblance in primary structure between part of the insulin ~ chain (amino acids 23-29: -Gly-Phe-Phe-Tyr-Thr-Pro-Lys-) and substance P (amino acids 3-9, in reverse: - G l y - P h e - P h e - G l n - G l n - P r o - L y s - ) . Although this resemblance between sequences aligned in opposite directions is unusual, it is of interest to note that 5 of the 6 conserved amino acids in this region of insulin are apparently 'identical' to those in the neuropeptide. A key question concerning these and similar polymeric forms relates to their possible occurrence in vivo. Although it has not, so far, been suggested that insulin fibrils normally occur, there is some evidence that fibrils of insulin (or a fragment or precursor form) occur, in pancreatic islets, in the extracellular amyloid deposits associated with normal aging, maturity onset diabetes or B cell adenomas [5, 11]. The mechanism of formation of these amyloid fibrils, which are in a B-pleated configuration [4], is not known although the insolubility and relative stability of insulin fibrils suggest that, once formed in pathological conditions, an accumulation of similar fibrils might occur. In contrast the apparent tendency in vitro of substance P (undecapeptide) fibrils to form and dissociate under more normal 'physiological' conditions suggests that substance P itself may be a less likely candidate for a constituent of the amyloid deposited in the nervous system, such as that found in senile plaques or blood vessels [5] unless particular agents are involved, pathologically, in stabilizing the fibril. It is however possible that where substance P is normally concentrated in the neuron at, for example, synthetic, transport, storage or release sites, fibrillary or similar polymeric forms may normally exist.

325

Current investigations are aimed at establishing whether fibrillary forms of substance P or a related peptide can be identified in nervous tissue. We are grateful for the advice and help of J.A. Edwardson, B.E. Tomlinson, R.H. Pain, A. Hendry and C.R. Snell, and also for the financial support of the Wellcome Trust. 1 Burke, M.J. and Rouguie, M.A., Cross-/3-protein structures. 1. Insulin fibrils, Biochemistry, 11 (1972) 2435-2439. 2 Candy, J.M., Oakley, A.E., Perry, E.K. and Perry, R.H., Existence in vitro of fibrillary aggregates of substance P, cholecystokinin octapeptide, somatostatin and related molecules, J. Physiol. (Lond.), in press (abstract). 3 Glenner, G.G., Eanes, E.D., Termine, J.D., Bladen, H.A. and Limke, R.P., The structural characteristics of some proteins having the properties of Congo red amyloid fibrils, J. Histochem. Cytochem., 21 (1973) 400 (abstract). 4 Glenner, G.G., Amyloid deposits and amyloidosis, New Engl. J. Meal., 302 (1980) 1283-1292. 5 Glenner, G.G., Amyloid deposits and amyloidosis, New Engl. J. Med., 302 (1980) 1333-1343. 6 Kedar, I., Ravid, M. and Sohar, E., In vitro synthesis of 'amyloid' fibrils from insulin calcitonin and parathormone, lsr. J. meal. Sci., 12 (1976) 1137-1140. 7 Pauling, L. and Corey, R.B., Configurations of polypeptide chains with favored orientations around single bonds: two new pleated sheets, Proc. nat. Acad. Sci. (Wash.), 37 (1951) 329-340. 8 Peters, A., Palay, S.L. and Webster, H. de F., The Fine Structure of the Nervous System, W.B. Saunders Co., Philadelphia, 1976. 9 Waugh, D.F., A fibrous modification of insulin. 1. The heat precipitate of insulin, J. Amer. Chem. Soc., 68 (1946) 247-256. 10 Waugh, D.F., Regeneration of insulin from insulin fibrils by the action of alkali, J. Amer. Chem. Soc., 70 (1948) 1850-1857. 11 Westermark, P., Grimelius, L., Polak, J.M., Larsson, L.-I., Van Noorden, S., Wilander, E. and Pearse, A.G.E., Amyloid in polypeptide hormone-producing tumors, Lab. Invest., 37 (1977) 212-215.