Cleavage of type VIII collagen by human neutrophil elastase

Biochimica et Biophysica Acta, 1139 (1992) 295-299 © 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00

295

BBADIS 61183

Cleavage of type VIII collagen by human neutrophil elastase Reinhold Kittelberger a, T. James Neale b, Kathy T. Francky c, Nicholas S. Greenhill d and Gary J. Gibson e,1 " Wallacecille Animal Research Centre, Upper Hurt (New Zealand), b Department of Medicine, Wellington School of Medicine, Wellington South (New Zealand), " Department of Orthopedic Surgery, Adelaide Childrens Hospital, Adelaide (Australia), d The Malaghan Institute, Wellington School of Medicine, Wellington (New Zealand) and e Department of Chemical Pathology, Adelaide Children's Hospital, Adelaide (Australia) (Received 4 February 1992)

Key words: Collagen type VIII; Collagen type X; Human neutrophil elastase

In this report, the susceptibility of type VIII collagen to human neutrophil elastase is compared to other extracellular matrix components. Type X collagen is degraded to specific fragments at a substrate to enzyme ratio of 5:1 after 20 h at room temperature, but type VIII collagen is almost completely degraded after only 4 h incubation at a substrate to enzyme ratio of 50 : 1 and partly degraded after only 15 min. Laminin, merosin and types I, III, IV and V collagen exhibit no susceptibility to neutrophil elastase under the latter conditions, while fibronectin is degraded.

Introduction

Type VIII collagen was first detected and isolated from bovine aortic endothelial cells, subsequently from other endothelial cell cultures and also from several tumor cell lines, from which it is secreted as three different molecular mass forms [1-3]. It is readily digested by pepsin to a 50 kDa fragment. After bacterial collagenase digestion, no peptides of molecular mass greater than 10 kDa can be detected [1,4]. Two structural models have been been proposed. According to the 'cassette model', the single chain is composed of three tandemly repeating cassettes of triple-helical sequences of 50 kDa, being flanked by short non-collagenous domains [5]. In an alternative model, the parental a-chains consist of a short collagenous sequence with globular domains at the ends [6]. This model has been confirmed by cloning and sequencing of the cDNA of rabbit type VIII collagen. [7]. A restricted tissue distribution of type VIII collagen has been reported by Kapoor et al. [8]. Recent studies clearly identified the presence of this collagen variant in blood vessels [9-11], where it is predominantly located in the subendothelium [9,10] and revealed a more widespread distribution of type VIII collagen [10]. Involvement of this

Correspondence to: R. Kittelberger, Wallaceville Animal Research Centre, PO Box 40063, Upper Hurt, New Zealand. 1 Present address: Bone and Joint Center, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, Michigan. 48202, USA.

collagen type in heart development [12] and increased levels in brain tumors [13] have been reported very recently. Neutrophil elastase cleaves type X collagen to produce a major enzyme-resistant fragment of 32 kDa similar in size to that produced by mammalian collagenase [14]. Yamaguchi et al. found striking similarities between the cDNA sequences of collagens type X and type VIII [7]. We have shown that, despite similarities in the primary structure, type VIII collagen is more susceptible to this proteinase than type X collagen. Materials and Methods

Materials Human collagens type I, III, IV, V, laminin, fibronectin and merosin were obtained from Telios Pharmaceuticals, San Diego, CA, USA. Human neutrophil elastase was purchased from Calbiochem, San Diego, CA, USA.

Preparation and Characterization of Collagens Type X Collagen. Avian type X collagen was prepared as described in Ref. 15. Briefly, collagen and precursors present in the pooled culture medium of chicken chondrocyte cell cultures were precipitated by the addition of ammonium sulfate to 30% saturation, isolated by centrifugation and redissolved in 0.1 M Tris-HCI buffer (pH 7.4) containing 0.4 M NaC1 (TrisNaC1). After digestion with chymotrypsin for 6 h at

296 room temperature, type X collagen was purified by differential salt precipitation. The culture matrix was extracted by digestion with pepsin in 0.5 M acetic acid at 4°C followed by extraction with Tris-NaCl buffer at 4°C. Type X collagen was isolated from both the pepsin and Tris-NaC1 extracts and purified by differential salt precipitations. Type VIII collagen. A mixture of type VIII and V collagen was isolated from ovine Descemet's membranes, as described in Ref. 16, by limited pepsin digestion and differential salt precipitations. Pure type V I I I collagen (50 kDa triple-helical fragment) was isolated by gel filtration chromatography of this mixture on Agarose 1.5m [16]. The purified collagens were characterized by SDSP A G E , E L I S A and immunoblotting [9,15].

Enzyme Digestions Equal volumes of collagen (10 /~g/10 /~1) or glycoprotein solutions and human neutrophil elastase (2 /~g/10/~1 or 0 . 4 / ~ g / 1 0 p.l) solution in 50 mM Tris-F,,_., (pH 8.0), 0.3 M NaCI, 10 m M CaC12 were mixed and incubated at room t e m p e r a t u r e for defined periods of time (see Results). Control digestions were performed by mixing the protein with buffer instead of elastase and incubating the mixture for the same time as the longest enzyme digestion. The reaction was stopped by addition of PMSF to 2 mM. Aliquots of 20 /~1 were mixed with 30 ~1 of sample buffer (120 mM Tris-HC1 (pH 6.9), 10% SDS, 10% glycerol, 2% /3-mercaptoethanol) and heated at 70°C for 15 min before application of 30/~1 of these mixtures to SDS-polyacrylamide gels.

SDS-PAGE Gel electrophoresis was performed on either 7.5% homogeneous polyacrylamide gels or on 4 - 2 0 % gradient gels (Mini Protean II Ready Gels, Bio-Rad, Richmond, CA, USA), using the buffer system described by Laemmli [17]. After sample application, gels were run for 30 min at 200 V and then either Coomassie- or silver-stained [18]. Results

Cleavage of ovine collagen type V I I I and of murine collagen type X by neutrophil elastase at substrate to enzyme ratios of 5 : 1 is compared in Fig. 1 on Coomassie-stained SDS-polyacrylamide gels. Type X collagen was degraded to specific fragments (smallest peptide 32 kDa) resistant to further digestion by 20 h. Under the same conditions, type V I I I collagen was rapidly and almost completely degraded, without the appearance of detectable protein fragments. In order to visualize breakdown products of type V I I I collagen, digestion was performed at shorter time intervals and

Time/hours Type VHI Collagen 0

4

20

30

Type X Collagen 0 4 2(|

30

t

Elastase

Fig. 1. Sequential cleavage of ovine type VIII collagen and avian type X collagen by human neutrophil elastase at room temperature. The pepsin-digested fragments of both collagens were exposed to human neutrophil elastase at a substrate to enzyme ratio of 5:1 at the indicated times. Incubations were examined on a 7.5c;'~ SDS-po[yacrylamide gel and stained with Coomassie blue R 25(1. Tracks at time 0 (control incubations) show collagens withcmt the enzyme. incubated for 30 h at room temperature. with additional substrate to enzyme ratios (5:1 and 50: 1). To enhance detectability of cleavage products, silverstaining was used instead of Coomassie staining. The results are shown in Fig. 2. While the type VII1 collagen band disappears with continuation of the neutrophil elastase digestion, specific fragments appear quickly at substrate to enzyme ratios of 5 : 1, but also at a ratio of 50 : 1. Major fragment bands were of 21 kDa, 30 kDa and 42 kDa, as identified by globular molecular mass markers or approx. 15 kDa, 23 kDa and 30 kDa, when calculated as collagenous proteins. Surprisingly, the larger of these fragments become further degraded after prolonged incubation times while intact type VIII collagen is still present. A 21 kDa fragment of type VIII collagen is still visible after 4 h of incubation at a substrate to enzyme ratio of 5 : 1 on the silver stained gel (Fig. 2A) but not on the Coomassie stained gel (Fig. 1). A possible explanation for this discrepancy could be a more advanced digestion in Fig. 1, due to variability in the room temperature during incubation. Digestion of type X collagen by human neutrophil elastase over a time period of 4 h at substrate to enzyme ratios of 5 : 1 and 50 : 1 are shown in Fig. 3. At a ratio of 5 : 1, the type X collagen band decreases in intensity and a slightly smaller band appears and intensifies over the 4-h period. The lower molecular mass (32 kDa and 36 kDa)

297

f r a g m e n t s visible a f t e r 20 h d i g e s t i o n (Fig. 1) w e r e not s e e n a f t e r 4 h digestion. D i g e s t i o n at a s u b s t r a t e to e n z y m e r a t i o of 50 : 1 shows virtually no c h a n g e o f the type X c o l l a g e n b a n d intensity a n d t h e a b s e n c e of lower m o l e c u l a r m a s s f r a g m e n t s (Fig. 3). In o r d e r to c o m p a r e the susceptibility o f type V I I I c o l l a g e n to n e u t r o p h i l e l a s t a s e with t h e susceptibility o f o t h e r e x t r a c e l l u l a r m a t r i x c o m p o n e n t s to this enzyme, d i g e s t i o n s of several c o l l a g e n v a r i a n t s a n d t h r e e s t r u c t u r a l g l y c o p r o t e i n s w e r e p e r f o r m e d at a s u b s t r a t e to e n z y m e r a t i o o f 5 0 : 1 for 2 h at r o o m t e m p e r a t u r e . T h e s e a r e c o n d i t i o n s u n d e r which a c o n s i d e r a b l e b r e a k d o w n o f type V I I I c o l l a g e n occurs (see Fig. 2b). T h e results a r e p r e s e n t e d in Fig. 4. C o l l a g e n s type I, III, I V a n d V, l a m i n i n a n d m e r o s i n a r e not c l e a v e d u n d e r t h e s e c o n d i t i o n s , while f i b r o n e c t i n is d e g r a d e d .

Time/minutes 0

15

30

60

120

240

Mrl kDa

97664531-

~'--Elastase

21-

A

976645-

Timelminutes 0

15

30

60

120

240

31-

Mr/ kDa

21 -

"~'-Elastase B

976645-

31-

4- Elastase

2114A

Fig. 3. Sequential cleavage of type X collagen by human neutrophil elastase at room temperature. The pepsin-resistant fragment of type X collagen was exposed to human neutrophil elastase at a substrate to enzyme ratio of 5 : 1 (A) and at 50:1 (B) at the indicated times. Incubations were examined on 4-20% SDS-polyacrylamide gradient gel and visualized by silver-staining. Tracks at time 0 show type X collagen without added enzyme, but incubated for 240 min at room temperature. Elastase was only visible in (A), but the corresponding position is indicated in (B). Positions of globular molecular mass markers are shown at the left.

9766- ~

~

~

~

~

" Discussion

4531 . . . . . . . . . .

-~- Elastase

2114-

B

Fig. 2. Sequential cleavage of type VIII collagen by human neutrophil elastase at room temperature. The pepsin-digested fragment of type VIII collagen wasexposed to human neutrophil elastase at a substrate to enzyme ratio of 5 : 1 (A) and at 50 : 1 (B) at the indicated times. Incubations were examined on 4-20% SDS-polyacrylamide gradient gel and visualized by silver-staining. Tracks at time 0 show type VIII collagen without added enzyme, but incubated for 240 rain at room temperature. The last tracks show elastase without collagen. Elastase was only visible in (A), but the corresponding position is indicated in (B). Positions of globular molecular mass markers are shown at left.

C o m p a r e d to t h e f r a g m e n t a t i o n o f type X c o l l a g e n by h u m a n n e u t r o p h i l elastase, which is c o n s i s t e n t with p r e v i o u s r e p o r t s [14] (Fig. 1), t h e c l e a v a g e o f type V I I I c o l l a g e n o c c u r r e d m o r e r a p i d l y at a lower s u b s t r a t e to e n z y m e r a t i o a n d y i e l d e d lower m o l e c u l a r mass fragm e n t s (Figs. 2 a n d 3). T y p e V I I I c o l l a g e n was d e g r a d e d to specific i n t e r m e d i a t e f r a g m e n t s which w e r e f u r t h e r b r o k e n down a f t e r p r o l o n g e d e n z y m e t r e a t m e n t . A significant p r o p o r t i o n of t h e type V I I I c o l l a g e n b a n d still r e m a i n e d u n d i g e s t e d while t h e i n t e r m e d i a t e b a n d s a r e d i g e s t e d to s m a l l e r f r a g m e n t s (Fig. 2). A possible e x p l a n a t i o n is t h e existence of several d i f f e r e n t a c h a i n s [7,19], o n e of which m a y exhibit h i g h e r susceptibility to this p r o t e i n a s e .

298 Yamaguchi et al. have sequenced the a~(VII1) cDNA [7] and Mann et al. have sequenced the triple-helical domain of a purified second a-chain, ae(VllI) of bovine origin [19]. They found striking similarity between these two polypeptide chains and the a~-chain of type X collagen [7,19]. All three chains contain eight imperfections in the GIy-X-Y triplet structure in similar locations. Three of the al(X) chain imperfections, 5 of the al(VIII) chain imperfections and 4 of the a2(Vlll) chain imperfections contain a valine residue, which is likely to be vulnerable to neutrophil elastase [20]. The relatively high number of valine residues at triple-chain imperfections could explain why type VIII collagen is much more susceptible to human neutrophil elastase than type X collagen and why one a(VIII) chain may be more rapidly degraded than the other chain. Alternatively, once initial fragments have been formed, these may unwind, thus, exposing other valine-residues within helix interruptions and facilitating enhanced degradation. The difference in susceptibility of type VII1 and type X collagens to neutrophil elastase does not reflect species differences. Ovine and human type X collagen were shown to have a similar elastase susceptibility to the chick type X collagen (Gibson, Francki, Foster and Hopwood, data not shown). Incubation of collagens type I, III, IV and V with human neutrophil elastase under conditions which degraded type VIII collagen (2 h of incubation at substrate to enzyme ratios of 50 : 1 at room temperature), did not result in the digestion of these collagen types (Fig. 4). It has been reported that human neutrophil elastase does not attack type I collagen [21], which is consistent with our results. On the other hand t h e

degradation of type Ill collagen and of typc IV colla.gen [22] at comparable substrate to enzyme ratios has been shown, but these digestions were either performed over extended time periods (18 h for type I11 collagen) or at raised temperature (37°C for type IV collagen). Under the conditions applied, type VIII collagen exhibited much higher susceptibility to human neutrophil elastase than the other major collagen types. When the susceptibility of fibronectin, laminin and merosin (which is structurally related to laminin [23]), to human neutrophil elastase was examined, only fibronectin was degraded (Fig. 4). The degradation of fibronectin by this enzyme is in accordance with observations by Harlan et al. [24] In response to inflammatory stimuli, such as activated complement components, bacterial products, platelet-activating factor or leukotrienes, neutrophils adhere rapidly to the vascular endothelium, inducing endothelial cell lysis or detachment in vitro and edema in vivo [25]. Several mechanisms have been shown to produce activated neutrophil-derived endothelial injury, namely the generation of toxic oxygen products [26,27], the action of granular proteinases [28] and a mechanism independent of oxygen radicals and lytic enzymes [29]. The involvement of neutrophil elastase in endothelial cell detachment has been demonstrated in vitro [24,31] and it has been proposed that the underlying mechanism may be the digestion of endothelial surface proteins, such as fibronectin [24]. Here, we have confirmed the susceptibility of fibronectin to neutrophil elastase and have identified another extracellular matrix component, namely type VIII collagen, to bc highly susceptible to this enzyme. Both, fibronectin and

Type 1

Type III

Type IV

Type V

collagen

collagen

collagen

collagen

-

+

-

+

-I-

+

Fibronectin

Laminin

+

+

Mer~in _

+

Fig. 4. Comparison of the susceptibility of several extracellular matrix components to human neutrophil elastase. The proteins were either digested at an substrate to enzyme ratio of 50:1 for 120 rain at room temperature, indicated by (+), or incubated under the same conditions without the enzyme, indicated by (-). Incubations were examined on 4-20% SDS-polyacrylamidegradient gels and visualized by silver-staining.

Positions of globular molecular mass markers are shown at left.

299 type VIII collagen are components of the vascular endothelium [10,31,32]. Type VIII collagen is predominantly located in the subendothelial region of blood vessels and may be a constituent of the vascular endothelial basement membrane [9,10]. Laminin and type IV collagen, which are major components of basement membranes and type V collagen, which is closely associated with the vascular basement membrane [32], are not or are much less susceptible to neutrophil elastase. Therefore, we conclude that type VIII collagen is a major potential target for the lytic action of activated neutrophils in vivo. Recently, Iruela-Arispe and Sage [12] have identified a possible role for type VIII collagen during the morphogenesis of the heart. This collagen variant was found to be prominent during day 11 in the mouse embryo and stage 19 in the chicken embryo, but diminished in the newborn and adult. Therefore, the authors considered it plausible that type VIII collagen might facilitate cell migration and differentiation in the developing embryo. Due to the rapid turnover during embryogenesis transiently expressed collagens, such as type VIII collagen, must be more susceptible to collagenases and other proteinases than the interstitial collagens. This 'inherited' susceptibility to proteinases, e.g., to neutrophil elastase, may result in the disadvantageous susceptibility of the vascular endothelium to the lytic attack by activated neutrophils in the mature animal.

Acknowledgements We wish to thank Drs. J..J. Hopwood and B.K. Foster, Depts. Chemical Pathology and Orthopedic Surgery, Adelaide Children's Hospital, for their encouragement and provision of laboratory facilities. This research was supported by grants from the Health Research Council of New Zealand, the Adelaide Children's Hospital Research Trust and the National Health and Medical Research Council of Australia.

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