Chemical Changes in Elastin as a Function of Maturation - ACS

3 Chemical Changes in Elastin as a Function of Maturation ROBERT B. RUCKER and MICHAEL LEFEVRE

Downloaded by TUFTS UNIV on November 26, 2016 | http://pubs.acs.org Publication Date: May 28, 1980 | doi: 10.1021/bk-1980-0123.ch003

Department of Nutrition, University of California, Davis, CA 95616

This review focuses upon the post-translational modification and chemical changes that occur in elastin. Outlined are the steps currently recognized as important in the assembly of profibrillar elastin subunits into mature fibers. Descriptions of some of the proposed mechanisms that appear important to the process are also presented. It will be emphasized that from the standpoint of protein deterioration, elastin is a very novel protein. Under normal circumstances, the final product of elastin metabolism, the elastin fiber does not undergo degradation that is easily measured. Unlike the metabolism of many other proteins, deterioration or degradation is most evident biochemically in the initial stages of synthesis rather than as a consequence of maturation. Since the presence of crosslinks is an essential component of mature elastin, a section of this review also addresses important features of crosslink formation. For purposes of definition, we will use the following terms to designate the various forms of elastin. The term, n o n - c r o s s l i n k e d e l a s t i n , w i l l be used as a general d e s c r i p t i o n f o r p r o posed precursors to mature e l a s t i n that appear to be r a p i d l y modified during the initial stages o f e l a s t i c f i b e r formation. With respect to one o f these p r e c u r s o r s , the term, t r o p o e l a s t i n , has been used as a d e s i g n a t i o n f o r a n o n - c r o s s l i n k e d e l a s t i n p r e c u r s o r o f approximately 70,000 daltons (1). Since i t i s c u r r e n t l y the best c h a r a c t e r i z e d o f the n o n - c r o s s l i n k e d e l a s t i n s and i s used e x t e n s i v e l y by those f a m i l i a r with e l a s t i n , t h i s term w i l l be r e t a i n e d . E l a s t i n w i l l be used to designate the p r o t e i n i n i t s c r o s s l i n k e d form. T h i s term, however, i s at best o p e r a t i o n a l , s i n c e e l a s t i n i s only i s o l a t e d from t i s s u e s or c e l l c u l t u r e by procedures that would be o f f e n s i v e to most p r o t e i n chemists. As a component o f e x t r a c e l l u l a r m a t r i c e s , e l a s t i n i s extremely i n s o l u b l e and i n c l o s e a s s o c i a t i o n with many other e x t r a c e l l u l a r components (2). In order to remove these components, harsh treatments such as a u t o c l a v i n g , e x t r a c t i o n with a l k a l i or

0-8412-0543-4/80/47-123-063$05.00/0 © 1980 A m e r i c a n C h e m i c a l Society Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

64

CHEMICAL

DETERIORATION O F PROTEINS

organic a c i d s , e x t r a c t i o n with n o n - e l a s t o l y t i c enzymes, o r repeated e x t r a c t i o n with dénaturants are r e q u i r e d (1,3-7). Finally, when the term, e l a s t i c f i b e r i s used, the context w i l l a l s o be o p e r a t i o n a l , s i n c e e l a s t i c f i b e r s appear t o be composed o f s e v e r a l components ( e l a s t i n , c o l l a g e n and other f i b r i l l a r p r o t e i n s ) i n varying amounts (2).


Models f o r E l a s t i n and E l a s t i c i t y Before d e s c r i b i n g the major steps i n e l a s t i n b i o s y n t h e s i s , a general c o n s i d e r a t i o n o f the f u n c t i o n o f e l a s t i n i s p e r t i n e n t . When hydrated, e l a s t i n f i b e r s possess some o f the mechanical prope r t i e s o f polymeric rubbers (1,2,8,9). Skin, lung, ligaments, and major blood v e s s e l s contain a high concentration o f e l a s t i n , because o f the need i n these t i s s u e s f o r long-range, r e v e r s i b l e e x t e n s i b i l i t y (9). When e l a s t i c f i b e r s a r e observed, they a r e o f t e n found branched and fused i n the form o f a complex network. However, there i s a degree o f order i n t i s s u e s that are subjected to u n i d i r e c t i o n a l s t r e s s , such as the ligamentum nuchae o f ung u l a t e s , with the o r i e n t a t i o n o f the f i b e r s p a r a l l e l t o the d i r e c t i o n o f s t r e s s . In major blood v e s s e l s , e l a s t i c f i b e r s take on a l a m e l l a r arrangement i n the form o f c o n c e n t r i c sheets (10,11). S e r a f i n i - F r a c a s s i n i et a l . (12) and C l e a r y and C l i f f (13) have r e c e n t l y proposed t h a t e l a s t i n i n f i b e r s appears t o be present as filaments (15-25A i n diameter). S e r a f i n i - F r a c a s s i n i et a l . (12) argue that only the polymeric chains making up the f i l a m e n t s are c r o s s l i n k e d . There i s now s u f f i c i e n t data t o suggest that the filaments may contain a degree o f ordered s t r u c t u r e (1,8,12-14). The ordered s t r u c t u r e , however, i s q u i t e d i f f e r e n t from that f o r other s t r u c t u r a l p r o t e i n s , such as c o l l a g e n . The polymeric chains making up e l a s t i n appear to e x i s t i n the form o f 3 - s p i r a l s (3-turn s t r u c t u r e s ) separated every 6000-8000 daltons by α-helical segments c o n t a i n i n g a high concentration o f c r o s s l i n k s (14). T h i s i s an important p o i n t , s i n c e a h i g h l y ordered network f o r e l a s t i n would be incompatible with the entrophic i n t e r p r e t a t i o n o f an e l a s t i c r e c o i l ; p a r t i c u l a r l y the c l a s s i c a l view which r e q u i r e s that the c r o s s l i n k e d polymeric chains be i n random con formation. S e r a f i n i - F r a c a s i n i et a l . (12) argue that the f i l a ments possess such small diameters that they may a c t , however, as random chains. Morphological evidence suggests that the filaments are bonded by non-covalent i n t e r a c t i o n s t o form a r e t i c u l u m o r three-dimensional network as shown i n Figure 1. We f e e l that the filament model i s a t t r a c t i v e from a b i o l o g i c a l p o i n t o f view, s i n c e a degree o f molecular o r g a n i z a t i o n would be expected i n order t o form e l a s t i c f i b e r s that i n t e r a c t i n t i m a t e l y with the other filamentous elements that comprise the e x t r a c e l l u l a r matrix. It should be noted, however, that other models have been proposed f o r e l a s t i n . A d e s c r i p t i o n o f these models may be found i n r e views by Gosline (£) and Sandberg ( 1). c

Whitaker and Fujimaki; Chemical Deterioration of Proteins ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


3.

RUCKER AND LEFEVRE

Chemical Changes in Elastin

65

S p e c i a l f e a t u r e s o f e l a s t i n s t r u c t u r e are i t s unique amino a c i d composition (Table I) and amino a c i d sequences (Table I I ) . E l a s t i n i s one o f the most apolar p r o t e i n s i n nature. The high c o n c e n t r a t i o n o f v a l - p r o sequences as w e l l as the c r o s s l i n k s represented by amino acids such as desmosine (Des), isodesmosine (Ide) and l y s i n o n o r l e u c i n e (LNL) confer chemical s t a b i l i t y (Inc o n s i d e r a b l e e f f o r t over the l a s t few years has been d i r e c t e d t o ward sequencing p o r t i o n s o f e l a s t i n . Data from these s t u d i e s have provided much o f the b a s i s f o r the f i b r i l l a r model given i n Figure 1. I t i s now w e l l e s t a b l i s h e d that the polymeric chains o f e l a s t i n are composed o f a l t e r n a t i n g segments d i f f e r e n t i n amino a c i d composition (19-24). What are viewed as the extendable segments c o n t a i n repeating short peptide u n i t s c h a r a c t e r i z e d by t h e i r high content o f v a l i n e , p r o l i n e and g l y c i n e . Further, from data f o r amino a c i d sequences around some o f the c r o s s l i n k i n g amino acids (19,20,23) i t i s known that the peptides i n these regions f a l l i n t o two major c a t e g o r i e s based upon the amino a c i d residues f o l l o w i n g l y s i n e . In one group o f peptides, an aromatic amino a c i d r e s i d u e i s u s u a l l y found adjacent t o l y s i n e , whereas i n the other group o f peptides an a l a n i n e i s u s u a l l y found. This i s an important f i n d i n g , because, as w i l l be p o i n t e d out l a t e r , l y s i n e i s the p r e c u r s o r o f the c r o s s l i n k i n g amino acids i n e l a s t i n s . The presence o r absence o f an aromatic amino a c i d r e s i d u e adjacent to l y s i n e appears t o determine whether o r not i t w i l l be enzymatically modified. In a d d i t i o n , i n t e r a c t i o n s i n v o l v i n g aromatic r i n g s may f a c i l i t a t e the t r a n s f e r o f e l e c t r o n s i n the u l t i m a t e o x i d a t i o n o r r e d u c t i o n o f c e r t a i n c r o s s l i n k i n g amino a c i d s (19-21,23). Biosynthesis o f E l a s t i n E l a s t i c f i b e r s are u s u a l l y found i n t i s s u e s r i c h i n smooth muscle o r t i s s u e s c o n t a i n i n g f i b r o b l a s t s possessing some o f c h a r a c t e r i s t i c s o f smooth muscle c e l l s ( 4 ) . There i s a recent r e p o r t , however, that suggests e l a s t i n - l i k e p r o t e i n s may be s e c r e t e d from chondrocytes (25). When e l a s t i n i s s e c r e t e d , i t i s accompanied by other p r o t e i n s that appear t o be important t o i t s alignment i n t o f i b r i l s . One o f these p r o t e i n s i s r e f e r r e d t o as m i c r o f i b r i l l a r p r o t e i n ( c f . Table I, r e f . 2). When e l a s t i n i s s e c r e t e d , i t combines with the m i c r o f i b r i l l a r p r o t e i n t o form a complex which i s i n i t i a l l y r i c h i n the m i c r o f i b r i l l a r p r o t e i n . The r a t i o o f m i c r o f i b r i l l a r p r o t e i n to e l a s t i n , however, appears to decrease upon maturation ( 2 ) . Other p r o t e i n s a r e a l s o s e c r e t e d with m i c r o f i b r i l l a r p r o t e i n and e l a s t i n . I t i s now c l e a r that bound t o e l a s t i n i n i t s n o n - c r o s s l i n k form(s) i s a t r y p s i n - l i k e n e u t r a l p r o t e i n a s e (26). T h i s p r o t e i n a s e e f f e c t s



C H E M I C A L DETERIORATION O F PROTEINS

Figure 1. A model for elastin. The basis for the various figures are taken from ref erences 8 and 12-18. At 2000-3000 magnification, mature elastinfibersappear rope-like as shown in A. Thefibersare often branched and interconnected. At higher magnifica tion, the fibers appear amorphous and smaller interconnecting fibers are observed to bridge the largerfibers(B). At extremely high magnification the amorphousfibersappear to be made up of filaments containing systematically spaced striations (C). Models corresponding to the morphological features of the filaments have been proposed by Gotte (16). Three potential arrangements are given in D. The arrangements from left to right correspond to elastin in its stretched, relaxed and highly relaxed states. Figure Ε is our attempt to integrate the morphological features with chemical structure (14). The polymeric chains comprising elastin appear to be ordered in the form of β-spiral and α-helical segments. It is proposed that two polypeptide chains are cross-linked to comprise the filaments. The fihments in turn may be a reticulum of randomly crosslinked chains, if the bonding between the fihments arises from non-covalent forces (12). A possibility for the non-covalent cross-linking of the fihments is interactions involving calcium ions (14, 17, 18).


3.

RUCKER AND LEFEVRE

Chemical

Changes in

67

Elastin

Table I


AMINO ACID COMPOSITION (EXPRESSED AS RESIDUES PER 1000 TOTAL RESIDUES) OF TYPICAL MATURE ELASTIN, MATRIX COLLAGEN AND MICROFIBRILLAR PROTEIN PREPARATIONS.

Amino

Acid

Gly Ala Val Pro Hypro lie Leu Tyr Phe Thr Ser Asp+Asn Glu+Gln Met 1/2 Cys His Arg Lys Ides Des LNL Hexose % e

e

e

a

(G) (A) (V) (P) (P) (I) (L) (Y) (F) (T) (S) (D+N) (E+Q) (M) (C) (H) (A) CO

Crosslinked Elastin 0

332 228 138 117 16 25 60 6 29 10 10 8 16

-—

5 3 1 1.5 1.2 0

Common s i n g l e l e t t e r abbreviations

Bone Matrix Collagen

Microfibrillar Protein**

338 112 20 121 107 12 23 2 13 14 39 36 82 6

110 65 56 64

0

—

48 69 36 38 56 62 114 114 16 48 15 45 45

—

5 39 27

-N.C. 1

-N.C.

are given i n p a r e n t h e s i s .

I s o l a t e d from bovine ligamentum. Bovine bone matrix c o l l a g e n The l y s i n e - d e r i v e d c r o s s l i n k i n g amino acids are not c a l c u l a t e d (N.C). ^Composition taken from r e f . 2. S e e t e x t and Figure 4. e


68

C H E M I C A L DETERIORATION

O F PROTEINS

Table II COMMON AMINO ACID SEQUENCES IN NON-CROSSLINKED ELASTIN.


Repeating

units

Tetrapeptides:

GGVPGAVPGGVPGGVFFPGAG LGG LG

Pentapeptides:

YGAAGGLVPGAPGFG PGVGVPGVGVPGVGVPG(S)GV(P)GV(G)V PGV(G)(V)

Hexapeptides:

AAQFGLGPGIGVAPGVGVAPGV(G)VAPGVGV(A)PGVGVA PCX) I

Examples o f small t r y p t i c peptides c o n t a i n i n g A l a - and L y s - r i c h sequences^ Sequence AAAK AAK SAK APGK AK YGAK

Moles/mole p r o t e i n 6 6 2 2 1 2

'Amino a c i d sequences o f s p e c i f i c t r y p t i c peptides found i n p o r c i n e t r o p o e l a s t i n . When t e n t a t i v e assignments are given, the designations are i n parentheses. The common r e p e a t i n g u n i t s are u n d e r l i n e d . In c e r t a i n instances l i b e r t y was taken i n d e f i n i n g a common repeating u n i t when there was only amino a c i d d i f f e r e n c e . These sequences are common to the e x t e n s i b l e regions i n e l a s t i n . The t e t r a , penta o r hexa repeats appear t o correspond to 15 t o 25 percent o f the t o t a l residues i n the p r o t e i n . The source f o r the sequence data i s reference JL. 'Sequences commonly found i n the regions o f e l a s t i n that are e v e n t u a l l y i n v o l v e d i n c r o s s l i n k i n g ( c f . Figure 4 ) .


3.

RUCKER AND LEFEVRE

Chemical

Changes in

Elastin

69


cleavage o f the n o n - c r o s s l i n k e d forms o f e l a s t i n i n t o d i s c r e e t subunits ranging i n molecular weights from 12,000 t o 70,000 (26,27). In a d d i t i o n the enzyme, l y s y l oxidase, which i s i n v o l v e d i n the c r o s s l i n k i n g o f e l a s t i n i s a l s o s e c r e t e d ( c f . r e f s . 28-35 and the s e c t i o n on Formation o f S t a b l e E l a s t i n F i b r i l s ) . The exact form i n which n o n - c r o s s l i n k e d e l a s t i n i s s e c r e t e d from smooth muscle c e l l s i s yet t o be c l e a r l y d e f i n e d . F o s t e r e t a l . (36) have suggested that a n o n - c r o s s l i n k e d e l a s t i n (proe l a s t i n ) i s secreted from smooth muscle c e l l s i n a form that i s approximately 120,000 t o 140,000 d a l t o n s . They have suggested that p r o e l a s t i n i s cleaved t o s m a l l e r molecular weight forms o f non-crosslinked e l a s t i n . I t should be noted, however, that t h i s view i s not e n t i r e l y supported by data from other l a b o r a t o r i e s . There are two r e p o r t s on the use o f i s o l a t e d mRNA from chick a o r t a suggesting only a 70,000 d a l t o n n o n - c r o s s l i n k e d e l a s t i n i s the major product o f t r a n s l a t i o n (37> 3»§) · There i s a l s o a recent r e p o r t suggesting that a o r t i c mRMA t r a n s l a t e s a 200,000 d a l t o n p u t a t i v e e l a s t i n product (39). We have r e c e n t l y i s o l a t e d a nonc r o s s l i n k e d e l a s t i n from the aortas o f copper d e f i c i e n t c h i c k s t h a t appears to be 100,000 daltons (27). I t s amino a c i d composit i o n i s s i m i l a r t o that f o r t r o p o e l a s t i n (Table I I I ) . A major problem i n r e s o l v i n g these p o i n t s i s that the t r y p s i n - l i k e p r o t e i n a s e a s s o c i a t e d with e l a s t i n i s not e a s i l y denatured o r separated from the n o n - c r o s s l i n k e d forms o f e l a s t i n . The p r o t e i n a s e i s a l s o not r e a d i l y i n h i b i t e d by commonly used i n h i b i t o r s f o r t r y p s i n - l i k e p r o t e i n a s e s (26). In keeping with the concept o f s e v e r a l forms o f s o l u b l e e l a s t i n , Figure 2 o u t l i n e s the various steps which are envisioned to be i n v o l v e d i n the formation o f the e l a s t i n f i b r i l a s s o c i a t e d with the m i c r o f i b r i l l a r components. The process, a t l e a s t i n concept, i s not e n t i r e l y d i s s i m i l a r t o the p r o c e s s i n g and s y n t h e s i s o f c o l l a g e n f i b r i l s (40). Once r e l e a s e d from ribosomes, i t appears that n o n - c r o s s l i n k e d e l a s t i n i s i n c o r p o r a t e d i n t o f i b r i l s i n a matter o f minutes (27). Although i t i s not c l e a r what exact r o l e the p r o t e i n a s e ( s ) p l a y s , l i m i t e d p r o t e o l y s i s could act as s i g n a l s f o r other p o s t - t r a n s l a t i o n a l events, such as c r o s s l i n k i n g . A l t e r n a t e l y , p r o t e o l y s i s may c o n t r o l the net amounts o f e l a s t i c f i b e r s synthesized during given periods o f development. Nevertheless, unique with regard t o other examples where proteinases play a role i n protein regulation, e l a s t o l y t i c p r o t e i n a s e ( s ) appears t o f u n c t i o n i n normal development a t e a r l y steps i n e l a s t o g e n e s i s . I t i s o f i n t e r e s t t o note t h a t , t o date, no t r u e e l a s t i n a s e that r e a d i l y degrades mature e l a s t i n i n i t s c r o s s l i n k e d s t a t e has been i s o l a t e d from e l a s t i n - s e c r e t i n g c e l l s . Although the enzyme e l a s t a s e has been s t u d i e d e x t e n s i v e l y , one should keep i n mind that i t has only been i s o l a t e d from organs, such as the pancreas, i n v o l v e d i n d i g e s t i v e f u n c t i o n s and phagocytic c e l l s , such as macrophages and leucocytes. With respect t o f a c t o r s that cause s t i m u l a t i o n o f e l a s t i n s y n t h e s i s i n t i s s u e s , there i s some evidence t o suggest that



70


Figure 2. Synthesis of mature elastin fibers. Some evidence suggests the possibility for proforms to elastin that appear as the first products of transition. These products are cleaved to tropoelastin (27), which appears to combine with microfibriUar protein. Although post-translational events important to the synthesis of the microfibrillar protein have not been defined, it is clear that it is a major component on which is organized or assembled the profbriUar forms of elastin. Cross-linking is catalyzed by lysyl oxidase, a copper-requiring protein (30). Recent information on the elastin proteinase(s) involved in tropoelastolysis would suggest that proteolysis may also play a role in elastin fiber formation (24).


3.

RUCKER AND LEFEVRE

Chemical

Changes in

71

Elastin

Table I I I AMINO ACID COMPOSITION OF TROPOELASTIN AND A PUTATIVE TROPOELASTIN PRECURSOR (EXPRESSED AS MOLES PER 1000 MOLES OF AMINO ACID RESIDUE).


Amino A c i d

Tropoelastin Precursor

Tropoelastin

(90,000-100,000 daltons)

(72,000 daltons)

Lys His Arg Hypro Asp+Asn Thr Ser Glu+Gln Pro Gly Ala 1/2 Cys Val Met He Leu Tyr Phe

40 trace 12 8 15 17 13 23 121 315 165 0 169 0 16 44 12 25

42 0 6 8 5 10 8 13 127 335 175 0 177 0 18 54 11 30

c e r t a i n s t e r o i d hormones may a l t e r net synthesis (41-43). Also, there i s evidence that suggests i n c e r t a i n t i s s u e s e l a s t i n s y n t h e s i s occurs i n response t o mechanical a c t i v i t y (44). C e l l s t h a t produce e l a s t i n when grown on preformed i n s o l u b l e e l a s t i c f i b e r s w i l l s e c r e t e greater q u a n t i t i e s o f matrix p r o t e i n s i f the f i b e r s are s t r e t c h e d and r e l a x e d i n c u l t u r e than i f they are s t a t i o n a r y o r minced and a g i t a t e d . In lung, new e l a s t i n synthesis a l s o f o l l o w s the acute d e s t r u c t i o n o f e l a s t i n caused by i n h a l a t i o n o f e l a s t a s e o r papain (45). None o f these observations, however, has been put on a f i r m b a s i s at the molecular l e v e l . Formation o f Stable E l a s t i n

Fibrils

One o f the most important steps

in stabilizing

elastin



72

C H E M I C A L DETERIORATION OF PROTEINS

f i b r i l s i s the formation of c r o s s l i n k s . The c r o s s l i n k s r e s u l t from the o x i d a t i o n o f s p e c i f i c l y s y l r e s i d u e s . As mentioned above, these residues are located w i t h i n what appear to be defined c r o s s l i n k i n g regions i n the polypeptide chains making up the f i b r i l s (28-35,46,47). The enzyme r e s p o n s i b l e f o r the o x i d a t i o n i s l y s y l oxidase. The mechanism o f o x i d a t i o n i s probably s i m i l a r to an o x i d a t i v e deamination (28,^£) · Lysyl oxidase r e q u i r e s copper (30) and i s i n h i b i t e d by a f a m i l y of lathyrogens, such as 3 - a m i n o p r o p i o n i t r i l e (31). I t has been p u r i f i e d from a v a r i e t y of connective t i s s u e sources. However, there i s s t i l l no c l e a r d e f i n i t i o n regarding i t s s p e c i f i t y towards s p e c i f i c substrates (32,33). For example, e l a s t i n may serve as a substrate f o r l y s y l oxidases obtained from c o l l a g e n - r i c h sources that do not contain e l a s t i n (28-35). Recently i t has been demonstrated by Rayton and H a r r i s (30) that the r o l e o f copper, i n a d d i t i o n to i t s presumed r o l e as a c o f a c t o r , i s r e l a t e d to the i n d u c t i o n of l y s y l oxidase. Cycloheximide, but not actinomycin D, completely i n h i b i t s the incorporation of ^ C u i n t o l y s y l oxidase. They suggest that the mechanism may be s i m i l a r to the i n d u c t i o n o f f e r r i t i n by i r o n . It i s a l s o o f i n t e r e s t that when copper bound to serum p r o t e i n s i s added to c u l t u r e s o f minced a o r t a obtained from c o p p e r - d e f i c i e n t c h i c k s , the amount o f copper r e q u i r e d f o r i n d u c t i o n o f l y s y l oxidase i s one-tenth to one-twentieth o f that r e q u i r e d when copper s a l t s are added. Homogenizing the t i s s u e or incubating i t under ^ o r i n the c o l d blocks the appearance o f the enzyme. Under normal c o n d i t i o n s i t would appear that the enzyme i s secreted from c e l l s i n c l o s e a s s o c i a t i o n with i t s s u b s t r a t e . By conventional e x t r a c t i o n methods ( p h y s i o l o g i c a l b u f f e r s ) , most o f the l y s y l oxidase a c t i v i t y i s not r e l e a s e d from i n s o l u b l e conn e c t i v e t i s s u e f i b e r s (33,34). It i s only r e l e a s e d a f t e r extract i o n with dénaturants, such as urea. Further, i t i s d i f f i c u l t to handle i n s o l u t i o n because o f i t s tendency to form aggregates. L y s y l oxidase i s r i c h i n c y s t e i n e residues which may f a c i l i t a t e formation o f polymeric forms (33). It has a l s o been d i f f i c u l t to study the enzyme because n a t i v e substrates and i n h i b i t o r s o f the enzyme are extracted with i t i n t o urea and thus must f i r s t be d i s s o c i a t e d and removed before an estimation o f true a c t i v i t y can be obtained (44,45). Further, there are no w e l l - c h a r a c t e r i z e d substrates a v a i l a b l e f o r the r o u t i n e assay o f l y s y l oxidase. The standard assay i s a procedure described by P i n n e l l and Martin (31). The substrate i s prepared from embryonic chick aortas a f t e r c u l t u r e i n v i t r o i n the presence o f 4,5- or 6 - H - l y s i n e and i n h i b i t o r s o f endogenous l y s y l oxidase ( c f . Figure 3). With such an i l l - d e f i n e d substrate, and the requirement o f a p a r t i a l p u r i f i c a t i o n o f l y s y l oxidase before assay (46,47), the examination o f the enzyme's r o l e i n maturation or p a t h o l o g i c a l processes has been l e s s than q u a n t i t a t i v e . However, the a v a i l a b i l i t y o f the p u r i f i e d enzyme has allowed s e v e r a l i n v e s t i g a t o r s 1 1

3



3.

RUCKER AND LEFEVRE

Chemical

Changes in

Elastin

73

t o examine the i n t e r a c t i o n o f l y s y l oxidase with i t s s u b s t r a t e s . It i s now c l e a r that the enzyme only acts on c o l l a g e n and perhaps e l a s t i n when these p r o t e i n s are i n the form o f f i b r i l s (28>22~>]&>46). For example, only t r o p o c o l l a g e n serves as a s u b s t r a t e , not d i s s o c i a t e d subunits, such as c o l l a g e n α-chains (35). Following the o x i d a t i o n o f p e p t i d y l l y s i n e there i s l i t t l e evidence to date to suggest a r o l e f o r other enzymes i n c r o s s linking. The conversion o f p e p t i d y l l y s i n e to p e p t i d y l a l l y s i n e (Figure 3) appears to set the stage f o r the spontaneous formation o f c r o s s l i n k s . The major requirements are probably most r e l a t e d to the conformational s t a t e o f the p r o t e i n , the l o c a t i o n and j u x a p o s i t i o n o f l y s y l d e r i v a t i v e s , and what might be viewed as environmental f a c t o r s , e.g., oxygen t e n s i o n (28,29). For purposes o f t h i s manuscript, we wish to concentrate only on the steps l e a d i n g to the formation o f desmosines, amino acids found predominantly i n e l a s t i n . With respect to t h e i r formation, the f o l l o w i n g suggests t h e i r spontaneous formation from p e p t i d y l l y s i n e and the o x i d a t i o n product, p e p t i d y l a l l y s i n e . Narayanan et a l . (28 29) have shown that when p u r i f i e d l y s y l oxidase and n o n - c r o s s l i n e d e l a s t i n , s p e c i f i c a l l y t r o p o e l a s t i n , are incubated together, the desmosines are formed. Desmosine formation, how ever, only occurs at temperatures that favor f i b r i l l a r arrange ments o f t r o p o e l a s t i n . Subsequently, i t i s f e l t that the maturation o f non-crosslinked e l a s t i n i n t o c r o s s - l i n k e d e l a s t i n appears to i n v o l v e only two major steps, namely i n s o l u b l i z a t i o n through the formation o f f i b r i l s and f i x a t i o n o f the f i b r i l s by crosslinking. >:

To form the desmosines, three p e p t i d y l a l l y s i n e molecules and a molecule o f p e p t i d y l l y s i n e must condense. The steps i n condensation probably i n v o l v e the formation o f 1,2-dihydropyridines and 1,4-dihydropyridines as shown i n Figure 4 (19-24,46,48). Several kinds o f chemical evidence (46,48) suggest that the hydropyridines are e a s i l y o x i d i z e d under normal oxygen t e n s i o n to corresponding p y r i d i n i u m i o n s , such as the desmosines (isodesmosine or desmosine). The exact pathway by which the desmosines are formed, however, i s s t i l l not c l e a r . C u r r e n t l y , there are at l e a s t two views r e l a t e d to the mecha nism by which the desmosines are formed (19). These i n c l u d e the d i r e c t r e a c t i o n o f the s o - c a l l e d a l l y s i n e a l d o l ( c f . Figure 4) with dehydrolysinonorleucine to form desmosines, or a l t e r n a t i v e l y , the r e a c t i o n o f dehydromerodesmosine with an a l l y s i n e r e s i d u e . The f i r s t mechanism would r e q u i r e the formation o f the a l l y s i n e a l d o l and dehydrolysinonorleucine as i n t r a m o l e c u l a r c r o s s l i n k s . The second mechanism would r e s u l t from the stepwise a d d i t i o n o f two a l l y s i n e s and l y s i n e to form dehydromerodesmosine ( v i a Michael a d d i t i o n s ) and then condensation with a f o u r t h a l l y s i n e r e s i d u e . The major problem i n r e s o l v i n g these p o i n t s i s the d i f f i c u l t y o f sequencing around i n t r a - and i n t e r m o l e c u l a r c r o s s l i n k s i n a manner to provide d e f i n i t i v e information.



74


ala-lys — αία-αΐα-lys-tyr

I

I

H-C-H

=

H-C-H

Lysyl Oxidase / [Cu*] ala-allys-ala-ala-lys-tyr H' *0 C

Figure 3. Lysyl oxidase. The enzyme, lysyl oxidase, appears to seek out lysyl resi dues in alanyl- and lysyl-rich regions in the profibrilhr forms of elastin. The presence of an aromatic amino acid residue adjacent to lysine appears to block its oxidation. The product of oxidation is peptidyl a-aminoadipic-h-semialdehyde. Assays for the enzyme against elastin involve first the preparation of an elastin-rich pellet containing H-lysyl residues labeled in the 6 or 4,5 position. This is usually accomplished by incubating embryonic chick aortas in medium containing H-lysine plus β-aminopropionitrile (BAPN) to inhibit endogenous lysyl oxidase activity. BAPN is then removed having behind an elastin-rich residue in which the profibrilhr forms of elastin hbelled with H-lysine are only partially crosslinked. When lysyl oxidase preparations are added to this residue the release of tritium represents the assay for activity. It has also been demonstrated that tropoelastin, when incubated with lysyl oxidase, forms a-aminoadipich-semialdehyde and eventually crosslinks as shown in Figure 4. 3

3

3


3.

RUCKER AND LEFEVRE

Chemical

Changes in

Elastin

75


There i s s t i l l no way o f determining whether or not a given desmosine c r o s s l i n k s 1, 2, 3, or 4 polypeptide chains o f e l a s t i n . Based on model s t u d i e s , however, the most favorable arrangement would be expected i f only two chains are c r o s s l i n k e d together by a desmosine (19). T h i s extends from observations that p o l y a l a n y l r i c h peptides t y p i c a l l y favor α-helical conformations and that i t i s d i f f i c u l t to interconnect more than two polypeptide chains around any given desmosine. With regard to the other amino acids that could p o t e n t i a l y c r o s s l i n k e l a s t i n , the exact number o f dehydrolysinonorleucine, dehydromerodesmosine and a l l y s i n e a l d o l residues that are i n v o l v e d as i n t r a - or i n t e r m o l e c u l a r c r o s s l i n k s , and the extent to which these residues may be reduced to form s t a b l e c r o s s l i n k s i s not known. The

E l a s t i c F i b e r ; A l t e r a t i o n s During

Maturation

With the above overview o f e l a s t i c f i b e r formation, a t t e n t i o n may now be d i r e c t e d at changes which occur i n e l a s t i n upon matura t i o n o f the f i b e r . As mentioned above, the f a c t that mature e l a s t i n and other components o f the e l a s t i c f i b e r are i n s o l u b l e a f t e r c r o s s l i n k i n g d i c t a t e s that harse procedures have to be used i n order to i s o l a t e the p r o t e i n . Because o f t h i s , i t i s o f t e n d i f f i c u l t to determine whether or not one i s d e a l i n g with a pure e l a s t i n or a mixture o f other s t r u c t u r a l p r o t e i n components and elastin. For example, when e l a s t i n s as defined by a l k a l i i n s o l u b i l i t y are i s o l a t e d from matrix synthesized by c u l t u r e s o f smooth muscle c e l l s , i t has been observed that the composition o f t h i s m a t e r i a l i s a l t e r e d upon maturation (49). Whether the changes i n composition represent changes that are due to d i f f e r e n c e s i n e l a s t i n or merely i n d i c a t e d i f f e r i n g amounts o f other s t r u c t u r a l p r o t e i n s behaving as e l a s t i n during i s o l a t i o n has been d i f f i c u l t to c l a r i f y . Obviously d i f f e r e n c e s i n composition and the r a t h e r harsh i s o l a t i o n procedures r a i s e s e r i o u s questions when one i s a s s e s s i n g homogeneity. Further, when one examines c r o s s l i n k s i n aged t i s s u e s using harse techniques, i t i s a l s o necessary to ask whether or not any o f the changes observed are due to age-related events or the method employed. An example o f the l a t t e r i s reported by Barnes et a l . (50). They examined e l a s t i n from guinea p i g a o r t a that had been p r e v i o u s l y r a d i o c h e m i c a l l y l a b e l e d with C - l y s i n e . When t h i s product (obtained a f t e r a l k a l i - e x t r a c t i o n o f the aortas) was f u r t h e r t r e a t e d with b o i l i n g o x a l i c a c i d i n order to o b t a i n a s o l u b l e , but c r o s s l i n k e d product, "α-elastin", the r a d i o a c t i v i t y a s s o c i a t e d with the desmosines i n the s o l u b i l i z e d e l a s t i n was g r e a t e r than that i n the o r i g i n a l s t a r t i n g m a t e r i a l . The reason f o r t h i s i s probably r e l a t e d to the c o n d i t i o n s used i n the i s o l a t i o n , which forced the formation o f desmosine from i t s precursors i n a manner i n keeping with the scheme shown i n Figure 4. With respect to data on d i f f e r e n c e s i n the c r o s s l i n k i n g amino 1 4


C H E M I C A L DETERIORATION OF PROTEINS

R

Chemical Changes in Elastin as a Function of Maturation - ACS

Recommend Documents