Initial Characterization of Nephila ... - American Chemical Society

Chapter 6 Initial

C h a r a c t e r i z a t i o n o f Nephila Dragline 1

1

clavipes

Protein 2

2

1

C. M. Mello , Κ. Senecal , B. Yeung , P. Vouros , and David Kaplan

Downloaded by MONASH UNIV on January 11, 2016 | http://pubs.acs.org Publication Date: December 3, 1993 | doi: 10.1021/bk-1994-0544.ch006

1

Biotechnology Division, Natick Research, Development, and Engineering Center, U.S. Army, Natick, MA 01760 Department of Chemistry, Northeastern University, Boston, MA 02115 2

The molecular composition of the dragline fiber and the major ampullate gland protein from the golden orb-weaving spider, Nephila clavipes is not well defined. We report initial characterization of the major ampullate gland proteins and the dragline fiber including amino acid composition, SDS gel electrophoresis, peptide sequencing and N-terminal sequencing. Preliminary results have identified the presence of disulfide bonds within the dragline protein. Previously published cDNA sequence data of the spidroin I protein from the major ampullate gland of N. clavipes (1) corresponds with our sequence data presented below. S i l k i s produced by a variety of insects and spiders. The most extensively studied s i l k is obtained from the larvae of Bombyx mori. This domestic silkworm produces one type of s i l k , cocoon s i l k , at a specific stage in i t s l i f e c y c l e , during the f i f t h larval instar just before molting. Modified salivary glands are responsible for producing the s i l k which i s spun from the mouth. Silkworm s i l k is predominantly a proteinaceous fiber which contains two primary structural proteins or fibroins, a 325 kDa and a 25 kDa f i b r o i n present in equimolar ratios, and a family of glue-like sericin proteins (2,3). In contrast to the silkworm, the spider produces many different types of s i l k , some of which are available throughout their l i f e c y c l e . Each type of spider s i l k originates from a different set of glands. The major ampullate gland produces the structural s i l k s for the orb frame, r a d i i and dragline; the flagelliform

0097-6156/94/0544-0067$06.00/0 © 1994 American Chemical Society In Silk Polymers; Kaplan, David, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


68

SILK POLYMERS: MATERIALS SCIENCE AND BIOTECHNOLOGY

gland produces the v i s c i d s i l k f o r prey capture; the aggregate gland synthesizes an adhesive s i l k ; the minor ampullate gland i s r e s p o n s i b l e f o r the support f i b e r s of the orb web; the c y l i n d r i c a l gland produces the cocoon s i l k ; the a c i n i f o r m gland produces the s i l k f o r wrapping captured prey; and the p i r i f o r m gland produces attachment s i l k s t o couple t o environmental s u b s t r a t e s (4). Thus, s p i d e r s i l k s represent an e v o l u t i o n a r i l y t a i l o r e d s e t of p r o t e i n s t o meet s p e c i f i c f u n c t i o n s . The mechanical p r o p e r t i e s of s i l k s and d e t a i l s of t h e i r secondary and t e r t i a r y molecular arrangements can help us r e s o l v e longstanding q u e s t i o n s concerning the s t r u c t u r e / f u n c t i o n r e l a t i o n s h i p s of repetitive structural proteins. The work presented here w i l l focus on the major ampullate gland of the s p i d e r , Ν. clavipes, and i t s d r a g l i n e p r o t e i n f i b e r . The major ampullate gland c o n t a i n s t h r e e d i s t i n c t regions, the p o s t e r i o r r e g i o n where s i l k i s synthesized i n the l i n i n g of the gland by e p i t h e l i a l c e l l s , the middle r e g i o n f o r the storage of p r o t e i n , and the a n t e r i o r r e g i o n which leads t o the s p i n n e r e t where the p r o t e i n i s spun i n t o a f i b e r . E p i t h e l i a l c e l l s l i n i n g the p o s t e r i o r r e g i o n of the ampullate gland of the s p i d e r , Araneus sericatus, have a r a d i a l microtubule system which provides f o r i n t r a c e l l u l a r t r a n s p o r t and s e c r e t i o n of f i b r o i n i n the form of s e c r e t o r y granules (5). E l e c t r o n microscopy i n d i c a t e s t h a t the microtubules run from the b a s a l t o luminar membranes i n the cytoplasm and s i l k " g l o b u l e s " have been observed a s s o c i a t e d with these m i c r o t u b u l e s . The p o l y p e p t i d e c h a i n a s s o c i a t i o n s , c o n c e n t r a t i o n s and conformations i n these granules are unknown. There i s l i t t l e data p u b l i s h e d on the molecular c h a r a c t e r i z a t i o n of the major ampullate gland p r o t e i n s . Candelas and her co-workers used r a d i o l a b e l l e d g l y c i n e and a l a n i n e t o demonstrated t h a t the major ampullate gland of N. clavipes produces a s i n g l e p r o t e i n when s t i m u l a t e d by mechanical d e p l e t i o n of the s i l k gland. T h i s p r o t e i n was shown t o migrate as a homogeneous band i n SDS g e l e l e c t r o p h o r e s i s with an estimated molecular weight of 320 kDa (6). However, recent cDNA sequence data from the major ampullate gland of N. clavipes suggests t h a t there may be two d i f f e r e n t p r o t e i n s comprising the d r a g l i n e f i b e r which are designated as s p i d r o i n I and s p i d r o i n I I (1,7). A d d i t i o n a l c h a r a c t e r i z a t i o n of the p r o t e i n s present i n the major ampullate gland and i n the d r a g l i n e s i l k f i b e r i s necessary t o determine the number of d i f f e r e n t s i l k p r o t e i n s comprising the d r a g l i n e f i b e r . Some of these experiments are r e p o r t e d here and i n c l u d e amino a c i d composition, SDS g e l e l e c t r o p h o r e s i s , p e p t i d e sequencing of the d r a g l i n e f i b e r and N-terminal sequencing.

In Silk Polymers; Kaplan, David, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

6. MELLO ET AL.

Characterization of Ν. clavipes Dragline Protein 69

M a t e r i a l s & Methods. Spiders. Spiders, N. clavipes, were c o l l e c t e d i n F l o r i d a and Panama and maintained i n the l a b o r a t o r y .


Recovery o f Dragline S i l k F i b e r s and Major Ampullate Glands. C o l l e c t i o n of the d r a g l i n e f i b e r by c o n t r o l l e d s i l k i n g o f t h e s p i d e r s was performed according t o Work and Emerson (8). D i s s e c t i o n s were c a r r i e d out i n s t e r i l e SSC b u f f e r (0.1M sodium c h l o r i d e and 0.015M sodium c i t r a t e , pH 6.4) c o n t a i n i n g 5mM phenylmethylsulfonyl f l u o r i d e (PMSF) and the major ampullate glands were p l a c e d i n l i q u i d nitrogen immediately f o l l o w i n g e x c i s i o n and s t o r e d a t -70°C. S o l u b i l i z a t i o n o f the Dragline F i b e r . D r a g l i n e f i b e r was s o l u b i l i z e d i n 9M L i B r and incubated a t 37°C overnight with shaking. O c c a s i o n a l l y an a d d i t i o n a l 8-12 hour incubation was necessary t o completely s o l u b i l i z e the f i b e r . The f i b e r was not r e a d i l y s o l u b l e i n 8M urea, however, L i B r s o l u b i l i z e d f i b e r s can be p l a c e d i n t o an 8M urea, 200mM acetate, pH 4.0 b u f f e r by u l t r a f i l t r a t i o n without s a c r i f i c i n g the s o l u b i l i t y . T h i s b u f f e r system was used f o r g e l e l e c t r o p h o r e s i s and the determination o f p r o t e i n concentration. Protein concentrations were determined with the b i c i n c h o n i n i c a c i d p r o t e i n assay k i t from Sigma. Chemical Cleavage o f the Dragline F i b e r w i t h N-Bromosuccinimide. Dragline p r o t e i n f i b e r was s o l u b i l i z e d i n 85% formic a c i d . A 10-fold excess (by weight) o f N-bromosuccinimide (NBS) was added immediately f o l l o w i n g s o l u b i l i z a t i o n . The r e a c t i o n mixture was l e f t on i c e f o r 1.5 hours, followed by room temperature incubation f o r 1.5 hours t o i n c r e a s e t h e rate of the reaction. An equal volume o f d r y acetone was added t o quench the r e a c t i o n and p r e c i p i t a t e t h e peptides. Peptides were p e l l e t e d by c e n t r i f u g a t i o n a t 16,000 χ g f o r 1 hour. The supernatant was removed, and the p e l l e t was d i s s o l v e d i n i n i t i a l mobile phase used i n the HPLC separation (see below). Most of the p e l l e t e d m a t e r i a l remained s o l u b l e i n the mobile phase. Any i n s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n and filtration. Peptide Mapping by HPLC. A Waters 625LC system equipped w i t h a photodiode array detector and a reverse-phase C-4, YMC column ( 250 χ 4.6 mm) was used f o r t h e s e p a r a t i o n o f d r a g l i n e s i l k peptides. The s o l v e n t system c o n s i s t e d o f A=water, B= a c e t o n i t r i l e , and C=100mM potassium phosphate, pH 3.0, where s o l v e n t C was kept a t 20% t o maintain a constant concentration o f 20mM potassium phosphate b u f f e r i n the mobile phase. A 45 minute l i n e a r gradient from 78% A t o 50% A separated t h e



70

p e p t i d e s . Peaks were c o l l e c t e d from consecutive runs and pooled f o r amino a c i d a n a l y s i s and N-terminal sequencing. S y n t h e t i c peptides, G l y ( A l a ) and ( A l a ) * H C l , used as standards were purchased from Sigma. 4


6

G e l E l e c t r o p h o r e s i s . An equal volume of a 2X sample b u f f e r (0.125M T r i s - H C l , pH 6.8, 20% g l y c e r o l , 4% SDS, 0.005% bromophenol blue) was added t o each sample c o n t a i n i n g 25-50μg of p r o t e i n i n 8M urea, 200mM a c e t a t e pH 4.0. For reducing c o n d i t i o n s , the 2X sample b u f f e r a l s o contained 4% 2-mercaptoethanol. A l l samples were incubated i n sample b u f f e r f o r a t l e a s t 1 hour a t room temperature followed by exposure t o a b o i l i n g water bath f o r 5 minutes. A Novex p r e c a s t discontinuous SDS polyacrylamide g e l , based on the c l a s s i c a l T r i s - g l y c i n e b u f f e r system of Laemmli was used (9) . Separations were c a r r i e d out a t a constant voltage (125 v o l t s ) u n t i l the bromophenol blue was 0.5-1.0 cm from the bottom of the gel. Gels were s t a i n e d with 0.1% coomassie blue R-250 i n 40% methanol and 10% a c e t i c a c i d and d e s t a i n e d with a 10% methanol, 7.5% a c e t i c a c i d s o l u t i o n .

Electroblotting. SDS-polyacrylamide g e l s of d r a g l i n e and major ampullate gland p r o t e i n s were e l e c t r o b l o t t e d onto p o l y v i n y l i d e n e f l u o r i d e (PVDF) membranes f o r amino a c i d a n a l y s i s and N-terminal sequencing. F o l l o w i n g e l e c t r o p h o r e s i s the g e l was washed with t r a n s f e r b u f f e r (lOmM 3-cyclohexylamino-propanesulfonic a c i d , pH 11.0 (CAPS), 5% methanol, and 0.005% SDS) f o r 30 minutes before b l o t t i n g . E l e c t r o p h o r e t i c t r a n s f e r was achieved a t a constant c u r r e n t of 200 mA f o r 4 hours. The membrane was a i r d r i e d and washed overnight i n M i l l i - Q water t o reduce background i n t e r f e r e n c e . Amino A c i d A n a l y s i s . The Waters Pico-Tag system was used f o r amino a c i d a n a l y s i s . B r i e f l y , the p r o t e i n s were d r i e d under vacuum, hydrolyzed i n constant b o i l i n g HC1 c o n t a i n i n g 0.5% phenol, n e u t r a l i z e d t o remove any r e s i d u a l a c i d and coupled t o p h e n y l i s o t h i o c y a n a t e (PITC) forming phenylthiocarbamyl (PTC) amino a c i d d e r i v a t i v e s . PTC-amino a c i d s were separated on a 15 cm Pico-Tag r e v e r s e phase column using a l i n e a r g r a d i e n t of sodium a c e t a t e b u f f e r ( c o n t a i n i n g 0.05% t r i e t h y l a m i n e (v/v) and 0.2Mg/ml of EDTA) and 60% a c e t o n i t r i l e ( c o n t a i n i n g 0.2Mg/ml of EDTA). A l l data were c o l l e c t e d and i n t e g r a t e d by VAXLAB and the Waters Expertease 860 software. N-terminal Sequencing. Automated Edman degradation of a l l p r o t e i n s and peptides was performed on an A p p l i e d Biosystems, Inc. Model 477A peptide sequencer equipped with an o n - l i n e PTH amino a c i d analyzer a t the C o r n e l l


6. MELLO ET AL.


U n i v e r s i t y , Biotechnology A n a l y t i c a l / S y n t h e s i s F a c i l i t y by Ted Thannhauser.


Results & Discussions. S o l u b i l i z a t i o n Procedures. Both d r a g l i n e f i b e r and major ampullate gland p r o t e i n were s o l u b i l i z e d i n 9M L i B r as d e s c r i b e d e a r l i e r . These samples were t r a n s f e r r e d t o 8M urea, 200mM acetate, pH 4.0 by u l t r a f i l t r a t i o n and analyzed by SDS-PAGE. Both samples are v i r t u a l l y i d e n t i c a l , migrating a t approximately 275 kDa. Gel e l e c t r o p h o r e s i s and amino a c i d composition (see Table I) of e l e c t r o b l o t t e d bands from the s o l u b i l i z e d f i b e r and e x t r a c t e d gland p r o t e i n suggests t h a t they are i n d i s t i n g u i s h a b l e . These r e s u l t s a l s o suggests t h a t complete s o l u b i l i z a t i o n of a l l p r o t e i n components w i t h i n the f i b e r has been achieved due t o the s i m i l a r i t y of the amino a c i d compositions of the f i b e r before s o l u b i l i z a t i o n and the 275 kDa e l e c t r o b l o t t e d band. The estimated molecular weight of 275 kDa i s lower than p r e v i o u s l y published r e s u l t s of 320 kDa f o r the g l a n d u l a r p r o t e i n (6). However, the molecular weight markers used by Candelas e t a l . were Β. mori s i l k f i b r o i n (estimated a t 400 KDa) and a myosin s u b u n i t (220 kDa) and the exact molecular weight has not been a c c u r a t e l y determined f o r the s i l k f i b r o i n of Β. mori. The use of d i f f e r e n t molecular weight markers and s l i g h t l y d i f f e r e n t e l e c t r o p h o r e t i c c o n d i t i o n s may account f o r the v a r i a t i o n i n molecular weight determinations. In a d d i t i o n , i t should be r e c o g n i z e d t h a t p r o t e i n s c o n t a i n i n g s i g n i f i c a n t percentages of g l y c i n e and a l a n i n e have been reported t o migrate anomalously during e l e c t r o p h o r e s i s , u s u a l l y r e s u l t i n g i n estimates higher than the a c t u a l molecular weight (10). N-Terminal Sequencing. Major ampullate gland p r o t e i n was e l e c t r o b l o t t e d onto PVDF membranes and N-terminal sequencing was done. I t was o f t e n d i f f i c u l t t o sequence more than t e n or twelve r e s i d u e s i n t o the p r o t e i n because of a c o n t i n u a l increase i n the a l a n i n e and g l y c i n e peaks from c y c l e t o c y c l e which was a t t r i b u t e d t o n o n - s p e c i f i c cleavage of the p r o t e i n . F i g u r e 1 r e v e a l s a l l of the sequence data obtained thus f a r from N-terminal sequence of major ampullate gland p r o t e i n . I t i s p r e d i c t e d t h a t these sequences are not N-terminal sequences, but n o n - s p e c i f i c cleavage products because the N-terminus i s blocked (personal communications w i t h Ted Thannhauser). N o n - s p e c i f i c cleavage may have occurred d u r i n g the i s o l a t i o n and e l e c t r o p h o r e t i c t r a n s f e r of the p r o t e i n s or during Edman degradation due t o a c i d or base h y d r o l y s i s . A r e p e t i t i v e motif has been i d e n t i f i e d and i s r e f l e c t e d i n sequence 1 of F i g u r e 1. T h i s repeat appears with 100% homology f o u r t e e n times i n



72 Table I .

Amino A c i d Composition of D r a g l i n e F i b e r and Major Ampullate Gland P r o t e i n 3

Amino D r a g l i n e Gold Electroblotted Electroblotted Acid Fiber Dragline Dragline Major Ampullate Fiber Fiber Gland 1


GLX SER GLY ARG ALA PRO TYR LEU

10.55 2.58 44.57 2.13 27.19 1.95 3.41 4.19

9.00 6.90 41.50 2.00 27.00 1.10 2.70 2.00

10.30 3.60 42.20 2.38 27.52 1.66 3.68 4.52

8.52 3.51 41.66 1.28 25.25 0.78 4.20 4.82

aAmino a c i d composition was determined by the Waters Pico-Tag system as d e s c r i b e d i n m a t e r i a l s & methods. Only the most abundant amino a c i d s are represented i n t h i s t a b l e . The g o l d d r a g l i n e f i b e r was d i r e c t l y a c i d hydrolyzed t o ensure a complete a n a l y s i s . Solubilized samples of both the d r a g l i n e f i b e r and major ampullate gland p r o t e i n were separated on an SDS p o l y a c r y l a m i d e g e l and e l e c t r o b l o t t e d onto PVDF membranes f o r amino a c i d a n a l y s i s . A l l of the samples are very s i m i l a r suggesting t h a t the s o l u b i l i z e d major ampullate gland p r o t e i n and the d r a g l i n e f i b e r are comprised of the same protein(s). Reference (11). 1

s p i d r o i n I and i n most cases, immediately p r e c e d i n g the r e p e t i t i v e motif i d e n t i f i e d below with the d r a g l i n e p e p t i d e sequences. Sequence 3 i n F i g u r e 1 i s s i m i l a r t o the r e p e t i t i v e motif of the d r a g l i n e p e p t i d e sequences presented below and sequence 4 has not been i d e n t i f i e d i n e i t h e r s p i d r o i n I or I I . Reduction and I n i t i a l P r o t e i n M o d i f i c a t i o n Experiments. F i g u r e 2 d e p i c t s the e f f e c t s of 2-mercaptoethanol on the f i b e r and gland p r o t e i n . When the s o l u b i l i z e d p r o t e i n s are reduced, two major bands occur with an apparent molecular weight of 195 and 220 kDa. The d r a g l i n e f i b e r i n the reduced s t a t e produces an a d d i t i o n a l band t h a t i s not present i n the gland p r e p a r a t i o n a t a molecular weight of 180 kDa. Amino a c i d a n a l y s i s of the reduced bands of the major ampullate gland p r o t e i n presented i n Table I I i n d i c a t e s t h a t the two most prominent bands are v e r y s i m i l a r . Unfortunately, the d r a g l i n e p r o t e i n would not t r a n s f e r w e l l enough t o achieve adequate amino a c i d composition of the reduced bands. As a r e s u l t , m o d i f i e d e l e c t r o b l o t t i n g p r o t o c o l s and a l t e r n a t i v e methods are being pursued f o r the determination of the amino a c i d composition and c o n t r i b u t i o n of c y s t e i n e / c y s t i n e r e s i d u e s i n the primary and secondary s t r u c t u r e s of the dragline protein(s).



6.

MELLO ET AL.


1.

Ala-Ala-GlY-GlY-Ala-ÇlY-Gln-GlV-Gly

2.

Ala-Ala-Ala-Ala-Gly-Ala-Gln-Gly-Gly-Tyr-Gly-Ala/Gly-Leu-Ala-AlaAla/Gly-Ala

3.

qiy- ?

4.

Thr-Thr/Gly-Gln-Ala/Gly-Ala-Ala-Ala-Gly-Ala-Gly/Ala

-G^n-GlY-Ala-gjlY/Ala-Arq

F i g u r e 1. N-terminal Sequencing of Major Ampullate Gland Protein. Sequences 1 and 2 represent a r e p e t i t i v e r e g i o n (underlined) which occurs w i t h i n the s p i d r o i n I sequence. A second repeat a l s o i n the s p i d r o i n I sequence i s r e f l e c t e d i n sequence 3 and has been found i n peptides generated from the d r a g l i n e f i b e r (see F i g u r e 5) . Sequence 4 does not occur i n any of the p u b l i s h e d data and t h e r f o r e must r e p r e s e n t a p o r t i o n of the p r o t e i n which i s c u r r e n t l y u n i d e n t i f i e d .

F i g u r e 2. Reduction of S i l k P r o t e i n s . The e f f e c t s of 2-mercaptoethanol on the d r a g l i n e f i b e r and major ampullate gland p r o t e i n ( s ) are depicted above. Novex molecular weight markers are used as standards. Lanes 1 and 2 represent the major ampullate gland p r o t e i n s i n the non-reduced and reduced states, respectively. Lanes 3 and 4 c o n t a i n the s o l u b i l i z e d d r a g l i n e f i b e r i n the non-reduced and reduced forms. Two major bands occur with an apparent molecular weight of 195 and 220 kDa f o r both the f i b e r and gland p r o t e i n (s) when reduced. The d r a g l i n e f i b e r i n the reduced s t a t e produces a band t h a t i s not present i n the gland p r e p a r a t i o n a t a m o l e c u l a r weight of 180 kDa.


74


Table I I . Amino A c i d Composition of Reduced Major Ampullate Gland P r o t e i n Amino Major Ampullate Acid Gland P r o t e i n


GLX SER GLY ARG ALA PRO TYR LEU

8.52 3.51 41.66 1.28 25.25 0.78 4.20 4.82

220 kDa Reduced Band 9.77 2.57 45.88 1.98 28.57 0.37 3.25 4.62

195 kDa Reduced Band 9.35 2.79 44.80 2.23 28.35 0.51 3.26 4.48

I n i t i a l experiments are c u r r e n t l y underway t o q u a n t i t a t i v e l y determine the content of c y s t e i n e and c y s t i n e r e s i d u e s i n each of the i n d i v i d u a l reduced and non-reduced s p e c i e s . Oxidation with p e r f o r m i c a c i d f o l l o w e d by amino a c i d a n a l y s i s r e v e a l e d approximately 0.36% c y s t e i n e i n the major ampullate gland p r o t e i n ( s ) . These r e s u l t s i n d i c a t e 7-9 p o s s i b l e c y s t e i n e r e s i d u e s and 3-5 d i s u l f i d e bonds per p r o t e i n c h a i n i f i t i s assumed t h a t the molecular weight i s 180-220 kDa. C y s t e i n e has not been found i n any of the p u b l i s h e d sequence data (1,7) or i n our p r o t e i n sequence data presented here. However, the s p i d r o i n I and s p i d r o i n I I cDNAs i d e n t i f i e d by Lewis e t a l . do not r e p r e s e n t complete sequences. Approximately 28-34% of the expected 180-220 kDa f u l l length s p i d r o i n I p r o t e i n i s r e f l e c t e d i n the cDNA sequence. A q u a n t i t a t i v e c o n t r i b u t i o n of the s p i d r o i n I I cDNA sequence can not be estimated because i t ' s f u l l length p r o t e i n product has not been i d e n t i f i e d and t h e r e f o r e an estimate of i t s molecular weight i s unknown. S p e c i f i c p r o t e i n m o d i f i c a t i o n s , such as, c y a n y l a t i o n with 2 - n i t r o - 5 thiocyano-benzoic a c i d t o s e l e c t i v e l y c l e a v e the p r o t e i n at c y s t e i n e r e s i d u e s may help us t o map the p o s i t i o n s of c y s t e i n e w i t h i n the d r a g l i n e p r o t e i n f i b e r . There i s evidence f o r a s i n g l e d i s u l f i d e l i n k a g e between the small and l a r g e f i b r o i n peptide chains i n silkworm cocoon s i l k (12) . I t has been suggested t h a t t h i s d i s u l f i d e bond, along with hydrophobic i n t e r a c t i o n s , may serve t o prevent premature p r e c i p i t a t i o n of the p r o t e i n i n the gland by i n t e r f e r i n g with the conformational t r a n s i t i o n which i s thought t o occur d u r i n g f i b e r formation (13). Thus, these i n t r a and i n t e r c h a i n i n t e r a c t i o n s p l a y a c r i t i c a l r o l e i n m a i n t a i n i n g s o l u b i l i t y of the p r o t e i n d u r i n g s y n t h e s i s , export, t r a n s p o r t and s e c r e t i o n o c c u r r i n g before the f i n a l s p i n n i n g process. These i n t e r a c t i o n s are probably v e r y important f o r s o l u b i l i t y of the s p i d e r s i l k


6. MELLO ET AL



p r o t e i n s as w e l l . Therefore, f u r t h e r a n a l y s i s i s necessary and c u r r e n t l y underway i n our l a b o r a t o r y t o d e f i n e the p o t e n t i a l r o l e of d i s u l f i d e l i n k a g e s i n these processes. Chemical D i g e s t . Peptides of the d r a g l i n e f i b e r were generated by chemical d i g e s t i o n with N-bromosuccinimide (NBS) . F i g u r e 3 represents a 16% g e l of the d i g e s t e d m a t e r i a l . These peptides were separated by r e v e r s e phase HPLC and f r a c t i o n s were c o l l e c t e d f o r amino a c i d a n a l y s i s and N-terminal sequencing. A t y p i c a l peptide map can be seen i n F i g u r e 4. Amino a c i d composition of a r e p r e s e n t a t i v e set of p u r i f i e d peptides i s presented i n Table I I I . A l l the peptides have very s i m i l a r amino a c i d compositions however, the a l a n i n e and g l y c i n e content i s low compared t o t h a t of the d r a g l i n e f i b e r . Based on the amino a c i d composition data i t appears t h a t the a l a n i n e and g l y c i n e r i c h sequences are being l o s t on the column or during d e s a l t i n g procedures p r i o r t o p i c o t a g a n a l y s i s . The sample (NBS peptide mixture i n Table I I I ) t h a t i s i n j e c t e d onto the column c l o s e l y resembles the amino a c i d composition of d r a g l i n e . P o l y a l a n i n e ( A l a ) H C l and G l y ( A l a ) were e

6

4

Table I I I . Amino A c i d Composition HPLC P u r i f i e d D r a g l i n e Peptides Amino Acid GLX SER GLY ARG ALA PRO TYR LEU

NBS

Peptide Mixture 8.60 3.55 51.20 2.29 21.99 1.24 1.14 4.02

A2 7.92 7.42 45.09 9.85 3.37 2.08 2.49 4.35

of

A10

A23

9.65 9.98 35.66 7.65 4.01 2.16 3.22 5.50

10.71 10.20 30.61 11.81 3.92 2.30 3.17 5.51

used as c o n t r o l s t o determine i f these p e p t i d e s would b i n d i r r e v e r s i b l y t o the HPLC column. The ( A l a ) ' H C l was e l u t e d from the column while the G l y ( A l a ) was r e t a i n e d . These r e s u l t s i n d i c a t e t h a t t h e r e may be some i r r e v e r s i b l e b i n d i n g of g l y c i n e and a l a n i n e r i c h sequences t o the column under the g r a d i e n t c o n d i t i o n s used. Further a n a l y s i s w i l l proceed v a r y i n g mobile phase c o n d i t i o n s i n an attempt t o achieve recovery of a l l peptides present i n the i n i t i a l NBS-digested mixture. Sequence data from three peptides i s presented i n F i g u r e 5. A l l sequence data i s very s i m i l a r r e v e a l i n g a 6

4



76


F i g u r e 3. NBS Digest of the Dragline F i b e r . Peptides generated by chemical d i g e s t i o n with N-bromosuccinimide are presented above i n a 16% SDS polyacrylamide g e l . Novex molecular weight markers were used as standards i n lanes M and M . The digested f i b e r can be seen i n the lane l a b e l l e d D. Many peptides have been produced r e p r e s e n t i n g a broad molecular weight range. L




6. MELLO ET AL.

I • (min)

0

1

' • ι

1

1

• ' ι ' ' ' ' ι ' ' ' ' ι ' ' 10

20

1

' ι • ' ' ' ι ' 30

1

1

1

ι

1

' ' ' ι

1

' ' ' I 40

F i g u r e 4. Peptide Map of the D r a g l i n e F i b e r . Peptides generated by chemical d i g e s t i o n with N-bromosuccinimide were separated by reverse phase HPLC. A typical p e p t i d e map i s presented above. The s e p a r a t i o n s were c a r r i e d out with a 45 minute l i n e a r g r a d i e n t from 78% A t o 50% A, where A=water, b = a c e t o n i t r i l e and c=100mm phosphate, pH 3.0 and s o l v e n t C was kept a t 20% t o maintain a constant c o n c e n t r a t i o n of 20mM potassium phosphate b u f f e r .


78

1-


? - ? -Leu-Gly-Ser-Gln-Gly-Ala-Gly-Gln- ? -Ala/Ser-Ser/Leu- ? - A l a -

? -Ser=^ÂTâ 2.

? -Ser-Leu-Gly-Ser-Gln-Gly-Ala- ? -Asp/Gly-

3.

? - ? -Leu- ? -Ser-Gln-Gly-Ala-Gly-Arg- ? -Ala-Leu

?

-Asp/Gly


Figure 5. N-Terminal Sequence of NBS-Dragline P e p t i d e s . Sequence data from three of the NBS-peptides are presented above. A l l sequence data i s very s i m i l a r and r e v e a l s a repeat (underlined) i n t h e s p i d r o i n I protein. A n a l y s i s of these sequences a l s o confirms t h a t s p e c i f i c cleavage i s o c c u r r i n g a t t h e C-terminal

repeat i n t h e s p i d r o i n I p r o t e i n (1) with t h e consensus sequence Tyr-Gly-Gly-Leu-Gly-Ser-Gln-Gly-Ala-GlyArg-Gly-Gly. T h i s r e p e t i t i v e sequence occurs with 100% homology t e n times i n s p i d r o i n I . A n a l y s i s o f these sequences a l s o r e v e a l s t h a t the chemical cleavage i s o c c u r r i n g a t the C-terminal of t y r o s i n e as expected. I t i s not s u r p r i s i n g t h a t a l l of these sequences a r e n e a r l y identical. T h i s consensus sequence occurs with an 80-90% homology a f t e r almost every t y r o s i n e r e s i d u e i n s p i d r o i n I. The second d r a g l i n e p r o t e i n i d e n t i f i e d by Himman and Lewis (7), s p i d r o i n I I , a l s o c o n t a i n s many t y r o s i n e r e s i d u e s y e t we have not i d e n t i f i e d any sequence o r amino a c i d composition data from any o f t h e p e p t i d e s analyzed t o suggest t h a t t h i s p r o t e i n i s present i n the d r a g l i n e f i b e r . I t i s p o s s i b l e however t h a t these sequences have not been recovered as i s t h e case w i t h t h e a l a n i n e and g l y c i n e r i c h sequences and continued a n a l y s i s of a d d i t i o n a l peptides i s being pursued. Conclusions. I n i t i a l c h a r a c t e r i z a t i o n of the d r a g l i n e f i b e r and t h e major ampullate gland from N. clavipes i s r e p o r t e d . The a b i l i t y t o maintain s o l u b i l i t y of the d r a g l i n e p r o t e i n i n 8M urea, 200mM acetate has provided us w i t h t h e means t o i n v e s t i g a t e the primary and secondary s t r u c t u r e o f the d r a g l i n e p r o t e i n by some c l a s s i c a l p r o t e i n chemistry techniques. A comparison of the d r a g l i n e p r o t e i n t o t h e major ampullate gland p r o t e i n by SDS-PAGE has r e v e a l e d t h a t they a r e v i r t u a l l y i d e n t i c a l , m i g r a t i n g as one band a t 275 kDa i n the non-reduced s t a t e and two bands a t 195 and 220 kDa i n the reduced s t a t e . A minor band occurs i n t h e reduced sample of the d r a g l i n e f i b e r a t 180 kDa which i s not present i n the reduced major ampullate gland p r o t e i n . I s o l a t i o n and c h a r a c t e r i z a t i o n o f t h i s band i s c u r r e n t l y underway i n our l a b o r a t o r y . In a d d i t i o n , N-terminal sequence data from t h e major ampullate gland and peptide sequences from NBS d i g e s t e d



6. MELLO ET AL.

Characterization of Ν. clavipes Dragline Protein

79

d r a g l i n e resemble r e p e t i t i v e sequences present i n t h e s p i d r o i n I p r o t e i n from the major ampullate gland o f Nephila clavipes. To date, we have not obtained any amino a c i d composition or sequence data t o confirm t h e presence o f s p i d r o i n I I i n the d r a g l i n e f i b e r . Q u a n t i t a t i v e c y s t e i n e a n a l y s i s of t h e major ampullate gland p r o t e i n r e v e a l s 0.36% c y s t e i n e . If i t i s assumed t h a t the molecular weight of t h e p r o t e i n i s 180-220 kDa, there are approximately 7-9 c y s t e i n e r e s i d u e s and 3-5 d i s u l f i d e bonds per p r o t e i n c h a i n . The absence of c y s t e i n e i n the p a r t i a l cDNA sequences p u b l i s h e d t o date (1,7) suggest t h a t t h e r e a r e unique domains w i t h i n the d r a g l i n e p r o t e i n which have not been i d e n t i f i e d . Therefore, f u r t h e r c h a r a c t e r i z a t i o n i s necessary t o determine the r o l e of c y s t e i n e / c y s t i n e r e s i d u e s i n the d r a g l i n e f i b e r .

Acknowledgements. Angela Choate, Kevin R e m i l l a r d and S c o t t Stockwell deserve r e c o g n i t i o n f o r c o l l e c t i n g t h e s p i d e r s . A s p e c i a l thanks i s extended t o Ted Thannhauser and Bob Sherwood from the C o r n e l l U n i v e r s i t y Biotechnology A n a l y t i c a l / S y n t h e s i s f a c i l i t y f o r amino a c i d a n a l y s i s and N-terminal sequencing. Literature Cited. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Xu, M. and Lewis, R. V. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 7120. T a s h i r o , Υ., Otsuki, Ε., and Shimadau, T. Biochim. Biophys. Acta, 1972, 257, 198. Sasaki and Noda, Biochim. Biophys. Acta, 1973, 310, 91. G o s l i n e , J . M., Demont, M. E. and Denny, M. W. Endeavour, 1986, 10, 37. B e l l , A. L. and P e a k a l l , D. B. J. Cell Biol. 1969, 42, 284. Candelas, G. C. e t . al. Biochem. Biophys. Res. Commun. 1983, 116, 1033. Himman, M. B. and Lewis, R. V. J. Biol. Chem. 1992, 267, 19320. Work, R. W. and Emerson, P. D. J . Arachnol. 1982, 10, 1. Laemmli, U. K. Nature, 1970, 227, 680. Beavis, R. C., Chait, B. T., C r e e l , H. S., Fournier, M. J . , Mason, T. L. and Tirrell, D. A. J. Amer. Chem. Soc. 1992, 114, 7584. Zemlin, J . C. 1968 Technical Report 69-29-CM, AD 684333, U.S. Army Natick L a b o r a t o r i e s , Natick MA Shimura, K., K i k u c h i , Α., Ohtomo, K., Katagta, Y. and Hyodo, A. J. Biochem. 1976, 80, 693. Lotz, B. and Colonna-Cesari, F. Biochimie, 1979, 61, 205.

R E C E I V E D June 29, 1993


Initial Characterization of Nephila ... - American Chemical Society

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