A model for interstitial collagen catabolism by mammalian collagenases

J. theor. Biol. (1991) 153, 585-602

A Model For Interstitial Collagen Catabolism by Mammalian Collagenases GREGG B. FIELDS

Department of Laboratory Medicine and Pathology and The Biomedical Engineering Center, University of Minnesota, Box 107, 420 Delaware Street S.E., Minneapolis, Minnesota 55455, U.S.A. (Received on 7 March 1991, Accepted in revised form on 12 June 1991) Mammalian collagenases cleave all three a chains of native, triple-helical types I, II, and III collagens after the Gly residue of the partial sequence Gly-[Ile or Leu]-[Ala or Leu] at a single locus approximately three-fourths from the amino terminus. There are an additional 31 sites in the triple-helical regions of types I, II, III, and IV collagens that contain the same partial sequence but are not hydrolyzed. A model has been developed to explain this remarkable specificity. The mammalian collagenase cleavage site in interstitial collagens is distinguished by: (a) a low side-chain molal volume-, high imino acid (>33%)-containing region that is tightly triple-helical, consisting of four G I y - X - Y triplets preceding the cleavage site, (b) a low imino acid-containing (< 17%), loosely triple-helical region consisting of four G I y - X - Y triplets following the cleavage site, and (c) a maximum of one charged residue for the entire 25 residue cleavage site region, which is always an Arg that follows the cleavage site in subsite P~ or P~. In addition, the high imino acidcontaining region cannot have an imino acid adjacent to the cleaved Gly-[Ile or Leu] bond (i.e. in subsite P2). Careful scrutiny of the 31 non-cleaved sequences reveals that none of those sites shares all of the characteristics of the cleavage site. The criterion of this model thus explain both cleaved and non-cleaved sequences in the triple-helical regions of types I, II, III, and IV collagen, and are supported by all known experimental and theoretical results on collagen catabolism and structure.

Collagens are the m a j o r structural proteins of all connective tissues, including skin, bone, tendon, cartilage, blood vessels, and b a s e m e n t m e m b r a n e (Bornstein & Traub, 1979; Miller & G a y , 1982). The most a b u n d a n t collagens are types I, II, and I I I , which are called interstitial collagens. Interstitial collagens consist of three peptide chains, called a chains, with unique primary sequences for each interstitial collagen. Over 90% o f the central portion of each a chain contains repeating G I y - X - Y triplets, where X is most often Pro and Y is most often H y p t . This repeating tripeptide sequence induces each chain to a d o p t a left-handed poly-Pro II helix. Three left-handed chains then intertwine to form a right-handed super-helix. This triple-helical c o n f o r m a t i o n makes interstitial collagens highly resistant to all proteinases except specific collagenases (Woolley, 1984). In all higher organisms, t Abbreviations used for amino acids follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature (1972). All amino acids are of the L-configuration. Alternative abbreviations used are: Hyp, 4-hydroxy-L-proline; Hyl, 5-hydroxy-L-lysine. 585 0022-5193/91/240585+ 18 $03.00/0

O 1991 Academic Press Limited

586

G.-B. FIELDS

interstitial collagens are catabolized by "mammalian" collagenases, such as those isolated from rabbit synovial cells (Fini et al., 1987) and human fibroblasts (Goldberg et al., 1986) and polymorphonuclear leukocytes (Hasty et al., 1990; Kn/iuper et al., 1990a-c), which have a characteristic and highly specific mode of action (BirkedalHansen et al., 1989). They cleave all three a chains of native types I, II, and III collagens at a single locus by hydrolyzing the peptide bond following the Gly residue of the partial sequences Gly-[Ile or Leu]-[Ala or Leu] located approximately three-fourths from the NH2 terminus (Miller et al., 1976b; Hofmann et al., 1978; Dixit et al., 1979). The basis for this specific hydrolysis has been attributed to any combination of five collagen structural properties at the collagenase cleavage site (Gross et al., 1980). These properties are (i) specific enzyme sensitive bonds, (ii) local sequence, (iii) distribution of "weak helix" triplets, (iv) distribution of Hyp, and (v) residue side-chain volume concentration. Hydrolysis of interstitial collagens occurs at a single locus containing a Gly-[Ile or Leu]-[Ala or Leu] sequence (Table 1) (Hofmann et al., 1978; Bornstein & Traub, 1979; Lang et al., 1979; Dixit et al., 1979; Seyer & Kang, 1981; Highberger et al., 1982). It is possible to identify a total of 31 different sites in all of the a 1(I) chains of chick, rat, and bovine (Wendt et al., 1972; Balian et al., 1972; Highberger et al., 1982; Glanville et aL, 1983), the a2(I) chains of chick and bovine (Dixit et al., 1977, 1979; Hofmann et al., 1978; Bornstein & Traub, 1979), the a l ( I I ) chain of bovine (Francis et al., 1978; Seyer et al., 1989), the a l ( I I I ) chains of human and bovine (Dewes et al., 1979; Lang et al., 1979; Seyer & Kang, 1981), the al(IV) chains of human, mouse, and bovine (Schuppan et al., 1982, 1984; Babel & Glanville, 1984; TABLE 1 S e q u e n c e o f 25 a m i n o acids surrounding the m a m m a l i a n interstitial collagens t

Collagen chain Chick al(1) Chick a2(l) Bovine ct1(I) Bovine a2(1) Human al(IlI) Bovine t~l(lII)

Sequence Gly-Pro-lle-Gly-Ala-Hyp-Gly-Thr-ProGly-Pro-Gln-GlyvTs-Ile-Ala-Gly-GlnArg-Gly-Val-Val-Gly-Leu-Hyp-Gly Gly-Ala-Ala-Gly-Pro-Hyp-Gly-Thr-HypGly-Pro-GIn-Gly775-lle-Leu-Gly-Ala-HypGly-Ile-Leu-Gly-Leu-Hyp-Gly Gly-Pro-Ala-Gly-Ala-Hyp-Gly-Thr-ProGly-Pro-Gln-GlyT75-Ile-Ala-Gly-GlnArg-Gly-Val-VaI-Gly-Leu-Hyp-Gly Gly-Ser-Ala-Gly-Pro-Hyp-Gly-Pro-HypGly-Pro-Gln-Gly775-Leu-Leu-Gly-AlaHyp-Gly-Phe-Leu-Gly-Leu-Hyp-Gly Gly-Ala-GIn-Gly-Pro-Hyp-Gly-Ala-HypGly-Pro-Leu-Gly775-Ile-Ala-Gly-lleThr-Gly-Ala-Arg-Gly-Leu-Ala-Gly Gly-Ala-Gln-Gly-Pro-Hyp-Gly-Ala-HypGly-Pro-Leu-Gly775-Ile-Ata-Gly-LenThr-Gly-Ala-Arg-Gly-Leu-Ala-Gly

f For each sequence, the P~ subsite is Glyvv5.

collagenase cleavage sites in

Reference Highberger et al. (1982) Dixit et al. (1979) Hofmann et al. (1978) Bornstein & Traub (1979) Seyer & Kang (1981) Lang et al. (1979)

INTERSTITIAL

COLLAGEN

CATABOLISM

587

M u t h u k u m a r a n et al., 1989; Saus et al., 1989), the a 2 ( I V ) chain o f h u m a n (Hostikka & Tryggvason, 1988), and the a 5 ( I V ) chain of h u m a n (Pihlajaniemi et al., 1990) collagens that contain G l y - [ I l e or L e u ] - [ A l a or Leu] sequences but are not cleaved by m a m m a l i a n collagenases in the native collagens (Table 2). A comparison o f the local sequence o f the eight a m i n o acids centered a r o u n d the G l y - [ I l e or Leu] b o n d s of each of the 31 non-cleaved sites with that of the cleaved sequence in the chick or bovine a l ( I ) chain was m a d e in order to identify amino acid substitutions that could potentially be responsible for the failure of these sites to be hydrolyzed by m a m m a l i a n collagenases. Based on this comparison, 13 octapeptides were synthesized, each o f which contained a single substitution relative to the otl(I) chain cleavage site sequence octapeptide G l y - P r o - G l n - G l y - I l e - A l a - G l y - G l n (Fields et al., 1987; Fields, 1988). Values of kcaJ K M for the hydrolysis o f each of the octapeptides by h u m a n fibroblast collagenase was determined by measuring the initial rates u n d e r first-order conditions (Fields et al., 1987; Fields, 1988; Netzel-Arnett et al., 1991). The most significant single substitution effect occurred when H y p was substituted for Gin in subsitet P_,, which decreased the rate of octapeptide hydrolysis by h u m a n fibroblast collagenase by 89%. In order to estimate the effects of multiple substitutions on the rates of hydrolysis of these sequences relative to that o f the a 1(I) sequence, it was assumed that the alterations in k c a J K M values produced by each individual amino acid substitution were independent and, thus, multiplicative. Two octapeptides with multiple substitutions were synthesized to test this assumption (Fields et al., 1987). The agreement between experimental and predicted values was satisfactory and the assumption of independence of substitutions a p p e a r e d reasonable (Fields et al., 1987; Fields & Van Wart, 1991; Netzel-Arnett et al., 1991). The predicted relative rates showed that the effect of these multiple substitutions ranged from changes of !.2-125% of the rate observed for the a l ( I ) sequence, with over half of the rates at least 10% of that of the known cleavage sequence (Fields et al., 1987; Fields & Van Wart, 1991; Netzel-Arnett et al., 1991). These results d e m o n strated that the sequence specificity of h u m a n fibroblast collagenase is not restrictive enough to account for the hydrolysis of native interstitial collagens at a single siteL The distribution o f " w e a k helix" triplets has received the most attention as the primary ,_'.nfluence o f collagenase specificity. The hypothesis had been put forth that there are "locally unstable" regions o f the triple-helix brought about by a local t The substrate subsites are designated according to the nomenclature of Schechter & Berger (1967). A sequence specificity study of human polymorphonuclear leukocyte (PMNL) collagenase, using the same series of octapeptides, resulted in the same general conclusion (Fields, 1988; Netzel-Arnett et al., 1991). However, agreement between the human fibroblast and PMNL collagenase studies should be considered to be only qualitative, as the activated PMNL collagenase species used for the specificity studies was generated from the 60 kDa proenzyme (Mookhtiar & Van Wart, 1990). A comparison of the known sequence of the 60 kDa PMNL procollagenase (consisting of the amino terminal 35 residues) (Mallya et al., 1990) with the complete sequence of the 75/85 kDa PMNL procollagenase (Hasty et al., 1990; Kniiuper et al., 1990a-c) shows that residues 1-35 of the 60kDa proenzyme are identical to residues 66-100 of the 75/85 kDa proenzyme. The 60 kDa PMNL procollagenase thus appears to be a degradation product of the 78/85 kDa PMNL procollagenase (Hasty et al., 1990; Michaelis et al., 1990). As the 75/85 kDa PMNL procollagenase has been shown to be activated by a complex, multi-step process (Kn~iuper et al., 1990a), generation of the physiologically relevent activated PMNL collagenase species is only ensured by the presence of the complete propeptide. The physiological significance of the activated species obtained from the 60 kDa PMNL procollagenase has not yet been established.

588

G.-B. F I E L D S TABLE 2

S e q u e n c e o f 25 a m i n o acids s u r r o u n d i n g potentially cleavable b o n d s in types I, II, I I I , and IV collagenst

Collagen chain Chick, bovine and rat a l ( I ) Chick al(1) Chick and bovine al(I) Chick and bovine a2(l) Chick a2(l) Chick a2(I) Chick a2(1) Bovine t~2(1) Bovine a2(1) Bovine a l ( l l ) Bovine a l ( l l ) Bovine ul(II) Bovine a l ( l l ) Bovine ct 1(III) Human a l ( l l l ) Human al(II1) Human al(III)

Sequence Gly-GIn- Hyp-Gly-Ala-Lys-Gly-Ala-AsnGly-Ala-Hyp-Glyz26-Ile-Ala-Gly-Ala Hyp-Gly-Phe- Hyp-Gly-Ala-Arg-Gly Gly-Ala-Hyp-Gly-Ser-Arg-Gly-Phe- HypGly-Ala-Asp-Gly322-Ile-Ala-Gly-ProLys-Gly-Pro- Hyp-Gly-Glu-Arg-Gly Gly-Glu-Arg-Gly-Pro- Hyp-Gly-Pro-MetGly- Pro- Hyp-Glyaz6-Leu-Ala-Gly-ProHyp-Gly-Glu-Ser-Gly-Arg-Glu-Gly Gly-Glu- Hyp-Gly- Lys- Hyp-Gly-Glu-LysGly-Asn-Val-Gly424-Leu-Ala-Gly-ProA.rg-Gly-Ala-Hyp-Gly-Pro-Glu-Gly Gly-Asp-VaI-Gly-Pro-Val-Gly-Arg-ThrGly-Glu-Gln-Gly745-Ile-Ala-Gly-ProHyp-Gly- Phe-Ala-Gly-Glu-Lys-Gly Gly-Thr- Hyp-Gly- Pro-Gin -Gly- lle- AlaGly-Ala-Hyp-Gly78 l- lie- Leu-Gly- LeuHyp-Gly-Gly-Arg-Gly-Glu-Arg-Gly Gly- Leu- Hyp-Gly-Ser-Arg-Gly-Glu-ArgGly- Leu- Hyp-Gly796- Ile-Ala-Gly-AlaThr-Gly-Gln- Hyp-Gly-Pro-Leu-Gly Gly-Val-Ala-Gly-Ser-VaI-Gly-Glu-HypGly- Pro- Leu-Glys08- Leu-Ala-Gty-ProHyp-Gly-Ala-Arg-Gly-Pro-Hyp-Gly Gly-Asp-Arg-Gly-His-Asn-Gly-Leu-GlnGly-Leu- Hyp-Gly943- Leu-Ala-Gly-HisHis-Gly-Asp-Gln-Gly-Ala-Hyp-Gly Gly- Ile- Hyp-Gly-Ala-Hyl-Gly-Ser-AlaGly- Ala-Hyp-Glyz26-Ile-Ala-Gly-AlaHyp-Gly-Phe-Hyp-Gly-Ala-Arg-Gly Gly-Pro- Leu-Gly- Pro-Hyl-Gly-Gln-ThrGly-Glu- Hyp-Gly259- Ile-Ala-Gly-PheHyI-Gly-Glu-Gln-Gly-Pro-Hyl-Gly Gly-Ala-Hyp-Gly-Ser-Arg-Gly-Phe-HypGly-GIn-Asp-Gly3zz-lle-Ala-Gly-ProHyl-Gly- Pro-Hyp-Gly-Glu-Arg-Gly Gly-Leu-GIn-Gly-Met-Hyp-Gly-Glu-ArgGly-Ala-Ala-Glys59-Ile-Ala-Gly-ProHyl-Gly-Asp-Arg-Gly-Asp-Val-Gly Gly- Pro-Arg-Gly-Glu-Arg-Gly-Glu-AlaGly-Ser-Hyp-GlY,s3-Ile-Ala-Gly-ProLys-Gly-Glu-Asp-Gly-Lys-Asp-Gly Gly-Asn-Ala-Gly-Ala-Hyp-Gly-Glu-ArgGly- Pro- Hyp-Glys t4- Leu-Ala-Gly-AlaHyp-Gly- Leu-Arg-Gly-Gly-Ala-Gly Gly- Pro-Leu-Gly- Ile-Ala-Gly-Ile-ThrGly-Ala-Arg-Gly7s4-Leu-Ala-Gly-ProHyp-Gly- Met- Hyp-Gly- Pro- Arg-Gly Gly-Glu-Arg-Gly-Pro- Hyp-Gly- Pro-AsnGly- Leu- Hyp-Glys29-Leu-Ala-Gly-ThrAla-GIy-Glu-Hyp-Gly-Arg-Asp-Gly

Reference Glanville et al. (1983) Highberger et al. (1982) Highberger et al. (1982) Bornstein & Traub (1979) Dixit et al (1979) Dixit et al. (1979) Dixit et al. (1979) Bornstein & Traub (1979) Bornstein & Traub (1979) Seyer et al. (1989) Seyer et al. (1989) Seyer et al. (1989) Francis et al. (1978) Dewes el al. (1979) Seyer & Kang (1981) Seyer & Kang (1981) Seyer & Kang (1981)

589

INTERSTITIAL COLLAGEN CATABOLISM TABLE 2 - - c o n t i n u e d Collagen chain Human and bovine al(III) Human al(IV) Mouse al(IV) Bovine a l(IV) Human, mouse and bovine al(IV) Human a2(IV) Human ct5(IV)

Sequence Gly-Asp-Lys-Gly-Glu-Gly-Gly-Ala-HypGly- Leu-Hyp-Gly616-Ile-Ala-Gly-ProArg-Gly-Ser-Hyp-Gly-Glu-Arg-Gly Gly- Ile-Hyp-Gly-Leu-Arg-Gly-Glu-HylGly-Asp-Gln-GlyloTo-Ile-Ala-Gly-PheHyp-Gly-Ser-Hyp-Gly-Glu-Hyl-Gly Gly-Ile-Hyp-Gly-Arg-Hyp-Giy-Asp-HylGly-Asp-Gln-GlymoTo- Leu-Ala-Gly-PheHyp-Gly-Ser-Hyp-Gly-Glu-Hyl-Gly Gly-lle-Xxx-Gly-Arg-Hyp-Gly-Ala-AspGly-Asp-Gln-Giyl070-Leu-Ala-Gly-PheHyp-Gly-Ser-Hyp-Gly-Glu-Hyp-Gly Gly-Asp-Hyl-Gly-Ser-Hyl-Gly-Glu/Asp-ValGly- Phe- Hyp-Glyl195-Leu-Ala-Gly-SerHyp-Gly-Ile-Hyp-Gly-Ser/Val-Hyl-Gly Gly-Phe-Pro-Gly-Ala-Pro-Gly-Thr-Val-GlyAla- Pro-Glyt3sg-Ile-Ala-Gly-Ile-Pro-Gin- LysIle-Ala-Ile-Gln-Pro Gly-Leu- Pro-Gly-Asn-lle-Gly-Pro-Met-GlyPro- Pro-Gly54t-Leu-Ala-Leu-Gln-GlyPro-Val-Gly-Glu-Lys-Gly

Reference Seyer & Kang (1981) Babel & Glanville (1984) Muthukumaran (1989)

et al.

Schuppan

(1984)

et al.

Muthukumaran (1989)

et al.

Hostikka & Tryggvason (1988) Pihlajaniemi et a/.(1990)

t For each sequence, the numbered Gly residue represents the potential P1 subsite.

deficiency ofimino acids, based on the decreased helicity of the 36 residue ot 1(I)-CB7 peptide isolated from chick skin (Highberger et al., 1979). The presence of a cleavable sequence in an unstable region could expose the scissle bond to the enzyme and account for the observed specificity (Miller et al., 1976a; Highberger et al., 1979; Gross et al., 1980; Rhy/inen et al., 1983). The collagenase cleavage site region in types I and III collagen (in solution) has been shown to be susceptible to attack by trypsin and a-chymotrypsin, suggesting a relaxation of helicity for this region. Native type I collagen was cleaved by trypsin and a-chymotrypsin to yield two fragments of virtually identical molecular weights to those produced by collagenase cleavage (Rhy/inen et al., 1983). These authors suggested that the reversible local relaxation of the collagen triple-helix in this region was due to c i s - t r a n s isomerization of peptide bonds involving Pro or Hyp residues (Rhy/inen et al., 1983). Native type III collagen was cleaved by trypsin and pronase at the Arg78o-Glyvsl bond, which is located five residues upstream from the collagenase cleavage site (Miller et al., 1976a; Birkedal-Hansen et al., 1985). Therefore, the collagenase cleavage site region in type III collagen is also believed to lack normal helicity, making it susceptible to cleavage by proteases other than collagenases. However, the local imino acid content of the collagen chains surrounding the non-cleavable sequences listed in Table 2 are not very different from that for the cleaved regions. Thus, this local deficiency per se cannot be the sole basis for the specificity of mammalian collagenases toward native collagens. The influence of Hyp on interstitial collagen cleavage by collagenase was examined by Miller et al. (1976b). Fragments of rat and bovine a l ( I ) and human a l ( I I I )

590

G.-B. FIELDS

chains that contained Gly-Ile-Ala or Gly-Leu-Ala sequences but were not cleaved in the native collagens by animal collagenases were compared to cleaved collagen sequences (Gross et al., 1974; Miller et al., 1976b). It was noted that the triplet immediately following the non-cleaved Gly-Ile-Ala and Gly-Leu-Ala sequences was Gly-X-Hyp. Since a G l y - X - H y p triplet was not seen following cleaved Gly-Ile/Leu-Ala sequences, it was proposed that collagenase recognition involved at least five residues on the carboxyl side of the cleavage site (subsites P]-P~), and that Hyp in the subsite P~ position prevented collagenase cleavage (Miller et al., 1976b). However, extensive sequencing of collagen chains has since proven that many non-cleaved Gly-Ile/Leu-Ala sequences do not have Hyp in subsite P~, while cleaved a2(I) chains do have Hyp in subsite P~ (see Tables 1 and 2). Thus, the location of Hyp in subsite P~ is not a definitive determinant of collagenase cleavage, although it may have some influence. As discussed earlier, Hyp in subsite P2 does have a significant detrimental effect on collagenase cleavage. The final factor which might be expected to influence the location of the coilagenase cleavage site is the distribution of bulky side-chain residues along the collagen chains. Since the structure of the collagen triple-helix is such that all residue side-chains are solvent exposed, it seems reasonable to assume a low molal volume is desired at one side of the cleavage site to permit enzyme accessibility. Gross et al. (1980) calculated the weighted side-chain molal volume per G l y - X - Y triplet for the rat-bovine composite ~ 1(I) chain. Their results showed that the collagenase cleavage site is preceded by a region of extremely low side-chain molal volume and followed by a region of extremely high side-chain molal volume. This pattern of low to high side-chain molal volume was unusual for the o~1(I) chain, and was thus assumed to be related to the accessibility of the cleavage site to collagenase. However, calculation of the average side-chain molal volume, using the values given by Bear (1952), per G l y - X - Y triplet for the four triplets on either side of the a 1(III) chain cleavage site in either human or bovine collagen (see Table 1) does not show a dramatic low to high side-chain molal volume pattern. The low to high side-chain molal volume pattern is therefore probably not a criteria for enzyme binding and/or hydrolysis, although a low side-chain molal volume preceding the cleavage site might be. A qualitative theory for collagenase specificity that combines several of the aforementioned collagen properties was developed by Brown et al. (1977). Utilizing a l ( I ) chain sequence data, they had proposed that collagenase recognizes the cleavage site based on a symmetrical arrangement of imino acids on both sides of a region of reduced helicity. A 16 amino acid region of reduced stability (containing only one imino acid) folds out of the collagen molecule. The six triplets on either side of this region show an "inverse symmetry" with regard to Pro and Hyp content. Collagenase was believed to consist of at least two identical subunits in order, like DNA restriction endonucleases, to recognize identical sites on either side of the "unwound" region in collagen simultaneously. One side of this region is cleaved preferentially due to a Pro residue in position 771, one of only four Pro residues found in the Y position in the ~1(I) chain. Unfortunately, both the symmetrical imino acid pattern and location of Pro in position 771 are features of the a l ( I )

INTERSTITIAL COLLAGEN CATABOLISM

591

chain cleavage site region exclusively, as a 2 ( I ) and a 1(III) chains do not contain these features (see Table 1). An " u n w o u n d " region of 16 amino acids is not consistent with c~2(I) and a 1(III) chain sequences, as there are several imino acids within this region for both chain types. In addition, mammalian collagenases have been shown to be a single polypeptide chain, not an associated dimer (Goldberg et al., 1986; Fini et al., 1987; Hasty et al., 1990; Kn/iuper et al., 1990a-c); thus, a comparison to the mode of action of D N A restriction endonucleases is not appropriate. All of the previously discussed hypotheses were based upon the cleavage or non-cleavage o f one type of collagen a chain. While some valid ideas are certainly present in these theories, none completely explain the specificity of mammalian collagenases for all known collagen c~ chain sequences. More importantly, these individual theoreis are completely unsatisfactory for explaining non-cleaved sequences. The approach has thus been taken of comparing the environments of the cleaved and non-cleaved sequences in interstitial (types I, II, and III) and type IV collagens to develop a model that accounts for the differential recognition of cleaved sequences by mammalian collagenases. The previously discussed peptide sequence specificity data clearly showed that the local conformation of collagen near the cleavage site, not the local sequence, must be a major factor in directing its own proteolysis. Welgus et al. (1982) have shown that h u m a n fibroblast collagenase makes multiple proteolytic cleavages in types I, II, and III denatured collagens (gelatins) at G l y - [ I l e or L e u ] - Y - G I y loci, where Y is many different residues. Thus, at least several loci (some of which are probably listed in Table 2) that are hydrolyzed by mammalian collagenases in denatured collagens are protected in the native, triple-helical collagen. In addition, the different kinetic parameters obtained for the human fibroblast collagenase hydrolysis of native collagens, gelatins, and octapeptides suggests that enzyme recognition and binding is modulated by the collagen triple-helix (Table 3) (Welgus et al., 1981, 1982; Fields et al., 1987, 1990b; Mallya et al., 1990; Netzel-Arnett et al., 1991). Specifically, the KM values are <1 /~M for the hydrolysis of type I

TABLE 3 K i n e t i c p a r a m e t e r s f o r s u b s t r a t e h y d r o l y s i s by h u m a n f i b r o b l a s t c o l l a g e n a s e

Substrate

kcat (hr -t)

KM (p.M)

Type I collagen (rat) Type I collagen (rat) Type I collagen (guinea-pig) Type I collagen (human) Type I collagen (human) al(I) gelatin (rat) a l(I) gelatin (guinea-pig) a2(I) gelatin (guinea-pig) Gly- Pro-Gln-Gly-Ile-Ala-Gly-GIn Gly- Pro-GIn-Gly-Leu-Ala-Gly-Gln

16 20 1700 44 53 24 230 750 730 970

0.83 0.90 0.90 0.82 0.80 9.8 7.0 3.7 3300 2800

k~t/ KM (/./.M-I hr-')

Reference

19 21.7 1890 54 66.8 2.4 33 203 0-22 0.35

Mallya et al. (1990) Welgus et al. (1981) Welgus et al. (1982) Mallya et al. (1990) Welgus et al. (1981) Fields et aL (1990b) Welgus et al. (1982) Welgus et al. (1982) Fields et al. (1987) Fields et al. (1987)

592

G.-B. FIELDS

collagens, 4-10 IZM for the respective gelatins, and > 1 mM for octapeptides of similar sequences (Table 3). The implication is that mammalian collagenases bind more efficiently to a cleavable sequence in native collagen than to an octapeptide or denatured collagen chain containing the same sequence. This triple-helical recognition feature appears to be applicable for mammalian collagenases only, and not for proteases in general. Studies utilizing triple-helical collagen model peptides (Fields et al., 1990a; Goli et al., 1991) have shown that the presence of a triple-helical region (seven Gly-Pro-Hyp triplets) prior to a nine-residue sequence containing a cleavable bond will inhibit non-specific protease (thermolysin) hydrolysis by 15-fold (Goli et al., 1991). The imino acid content of collagens has long been known to influence the thermal stability of the native molecule (Burge & Hynes, 1959; Privalov, 1982; Bhatnagar et al., 1988). More specifically, Pro in position X and Hyp in position Y stabilize the collagen triple-helix (Ramachandran et al., 1973; Sakakibara et al., 1973; Inouye et aL, 1976, 1982; Thakur et al., 1986; Germann & Heidemann, 1988). Therefore, it is of interest to examine the entire collagen sequence for imino acid content. Analysis of the rat-bovine composite otl(I) chain (Gross et al., 1980) shows that the mammalian collagenase cleavage site is marked by two distinct features: a high imino acid content prior to the cleaved bond, and low imino acid content after the cleaved bond. By comparing the sequences surrounding the cleavage sites in interstitial collagens (Table 1) for imino acid content of the four triplets prior to the P1 subsite Gly and four triplets after the P~ subsite Gly certain common patterns emerge. The region of four triplets that precedes the cleavage site contains a minimum of 33% imino acids (50% of the X + Y residues), with Pro always found in subsite P3, but no imino acid found in subsite P2. The four triplets that follow the cleavage site contain a maximum of 17% imino acids. In the two cases were this maximum of 17% imino acids occurs [bovine and chick a2(I) chains], the imino acids are not in neighboring triplets. In addition to imino acid content, analysis of charged residue density within the cleavage site regions shows a remarkably similar pattern. Within the same 25 amino acid region as discussed in the previous paragraph, a maximum of 1 (4%) charged residue is found, which is always Arg in subsite P~ or P~. This extremely low charge density is unusual, in that charged residues tend to "cluster" over several triplets in low imino acid containing regions in collagen (Drlz & Heidemann, 1986); therefore, the imino acid deficient region after the cleavage site would be expected to contain several charged residues. As previously mentioned, the region preceding the cleavage site has a low average side-chain molal volume. Calculations using the side-chain molal volumes of Bear (1952) reveal that, for the four triplets preceding the collagenase cleavage site (Table 1), the average side-chain molal volume per X / Y residue is <45 ml. A maximum of one residue is present with a side-chain molal volume >60 ml in the four triplets preceding the collagenase cleavage site. Based on the results described above, a model for the mammalian collagenase cleavage site in interstitial collagens has been developed (Fig. 1). The four triplet region that precedes the scissile bond is rich in imino acids, with the exception that

INTERSTITIAL

COLLAGEN

Loose triple-helix

Tight triple-helix

Collaqen

I

I I

I

I

I

,

I-- Pro --

593

CATABOLISM

[0o] Leu

L_

I

[ -- Gly

l',e ] [ A,o ] I Leu -- Leu -- Gly -- [

I

I

I

I

Collogenose FIG. 1. Model of the mammalian collagenase cleavage site in interstitial collagens. The four triplet region that precedes the scissile (Gly-Ile/Leu) bond is rich in imino acids, and has a low average side-chain molal volume. The four triplet region that follows the scissile bond is imino acid deficient. The overall 25 amino acid residue region is hydrophobic, containing <5% charged residues.

subsite P2 cannot contain an imino acid, and the average side-chain molal volume per X~ Y residue is <45 ml, with only one side-chain of molal volume >60 ml. This region of the triple-helix can thus be regarded as thermally stable, or "tightly triple-helical", with few bulky side-chains. The four triplet region that follows the scissile bond is deficient in imino acids, and can thus be regarded as less thermally stable, or "loosely triple-helical". This overall 25 amino acid residue region is also extremely hydrophobic, containing < 5% charged residues. It is reasonable to assume that the tightly triple-helical region serves as a recognition feature and binding site for mammalian collagenases, while the loosely triple helical region allows for enzyme access to and hydrolysis of the Gly-[Ile or Leu] bond. It is important to determine if the criteria of this model explain why certain sequences in collagen that contain potentially cleavable bonds are not cleaved. Table 2 shows the sequences of the 3 1 potentially cleavable, but non-cleaved, regions in types I, II, III, and IV collagens. Each of these sequences deviates from the cleavage site model as follows: (1-3) Chick, Bovine and Rat a l ( I ) Gly226-Ile: There are only 17% imino acids preceding the potential cleavage site, the two imino acids following the potential cleavage site are in neighboring triplets, Hyp is found in subsite P2, and a charged residue is found preceding the potential cleavage site (Lys in subsite Ps). (4) Chick a 1(I) Gly322-Ile: There are only 17% imino acids preceding the potential cleavage site, there are two side-chains of molal volume >60ml preceding the potential cleavage site, there are 25% imino acids following the potential cleavage site, and five charged residues are found in the overall region. (5, 6) Chick and Bovine a 1(I) Gly826-Leu: The average side-chain molal volume per X~ Y residue preceding the cleavage site is 52.2 ml (with two side-chains of

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molal volume >60 ml), the two imino acids following the potential cleavage site are neighbors (Pro in subsite P~, Hyp in subsite P~), Hyp is found in subsite P2, and five charged residues are found in the overall region. (7, 8) Chick and Bovine cr2(I) Gly424-Leu: There are only 17% imino acids preceding the potential cleavage site, the average side-chain molal volume per X~ Y residue preceding the cleavage site is 54.0 ml (with two side-chains of molal volume >60 ml), there are 25% imino acids following the potential cleavage site, and six charged residues are found in the overall region. (9) Chick a2(I) Gly745-Ile: There are only 8.3% imino acids preceding the potential cleavage site, the average side-chain molal volume per X~ Y residue preceding the cleavage site is 50-4 ml, and two imino acids following the potential cleavage site are neighbors (Pro in subsite P~, Hyp in subsite P~), and five charged residues are found in the overall region. (10) Chick a2(I) GlyTs~-Ile: There are only 25% imino acids preceding the potential cleavage site, Hyp is found in subsite P2, and three charged residues are found following the potential cleavage site. (11) Chick a2(I) Gly796-Ile: There are only 17% imino acids preceding the potential cleavage site, the average side-chain molal volume per X / Y residue preceding the cleavage site is 57.1 ml (with four side-chains ofmolal volume >60 ml), the two imino acids following the potential cleavage site are in neighboring triplets, Hyp is found in subsite P2, and three charged residues are found preceding the potential cleavage site. (12) Bovine a2(I) Glysos-Ile: There are only 17% imino acids preceding the potential cleavage site, there are 33% imino acids following the potential cleavage site, and a charged residue is found preceding the potential cleavage site (Glu in subsite P6). (13) Bovine a2(I) Gly943-Leu: There are only 8.3% imino acids preceding the potential cleavage site, the average side-chain molal volume per X~ Y residue preceding the cleavage site is 55.7 ml (with three side-chains of molal volume >60 ml), Hyp is found in subsite P2, and six charged residues are found in the overall region. (14) Bovine a l ( I I ) Gly226-Ile: There are only 17% imino acids preceding the potential cleavage site, there are two side-chains of molal volume >60 ml preceding the cleavage site, the two imino acids following the potential cleavage site are in neighboring triplets, Hyp is found in subsite P2, and two charged residues are found in the overall region. Of the two charged residues, Hyl in subsite Ps is glycosylated (Seyer et aL, 1989), increasing the average side-chain molal volume preceding the cleavage site. (15) Bovine a l ( I I ) Gly259-Ile: There are only 25% imino acids preceding the potential cleavage site, the average side-chain molal volume per X~ Y residue preceding the cleavage site is 52-6 ml (with two side-chains of molal volume >60 ml), Hyp is found in subsite P2, and five charged residues are found in the overall region. Of the five charged residues, the three Hyl residues (in subsites Ps, P~, and P'~t) are glycosylated (Seyer et aL, 1989), increasing the average side-chain molal volume preceding the cleavage site.

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(16) Bovine a l ( I I ) Gly3n-Ile: There are only 17% imino acids preceding the potential cleavage site, the average side-chain molal volume per X / Y residue preceding the cleavage site is 47.7 ml (with two side-chains of molal volume >60 ml), there are 25% imino acids following the potential cleavage site, and five charged residues are found in the overall region. Of the five charged residues Hyl in subsite P5 is glycosylated (Seyer et al., 1989), increasing the average side-chain molal volume preceding the cleavage site. (17) Bovine a l ( I I ) Gly559-IIe: There are only 8.3% imino acids preceding the potential cleavage site, the average side-chain molal volume per X / Y residue preceding the cleavage site is 50.1 ml (with three side-chains of molal volume >60 ml), and six charged residues are found in the overall region. Of the six charged residues, Hyl in subsite P5 is glycosylated (Francis et al., 1978), increasing the average side-chain molal volume preceding the cleavage site. (18) Bovine a l ( I I I ) Gly283-Ile: There are only 17% imino acids preceding the potential cleavage site, the average side-chain molal volume per X / Y residue preceding the cleavage site is 48-5 mi (with two side-chains of molal volume >60 ml), Hyp is found in subsite P2, and nine charged residues are found in the overall region. (19) Human ~1(III) Gly~4-Leu: There are only 25% imino acids preceding the potential cleavage site, Hyp is found in subsite P2, and two charged residues are found preceding the potential cleavage site. (20) Human a l ( I I I ) GlyTa4-Leu: There are only 8.3% imino acids preceding the potential cleavage site, the average side-chain molal volume per X / Y residue preceding the cleavage site is 50.3 ml (with four side-chains ofmolal volume >60 ml), there are 33% imino acids after the potential cleavage site, and a charged residue is found preceding the potential cleavage site (Arg in subsite Pz)(21) Human a l ( I I I ) Gly829-Leu: The average side-chain molal volume per X~ Y residue preceding the cleavage site is 51-9 ml (with two side-chains of molal volume >60 ml), Hyp is found in subsite P2, and five charged residues are found in the overall region. (22, 23) Human and Bovine a l ( I I I ) Gly6,6-Ile: There are only 17% imino acids preceding the potential cleavage site, there are two side-chains of molal volume >60 ml preceding the cleavage site, the two imino acids following the potential cleavage site are in neighboring triplets, Hyp is found in subsite P2, and six charged residues are found in the overall region. (24) Human al(IV) Gly~oTo-Ile: There are only 8-3% imino acids preceding the potential cleavage site, the average side-chain molal volume per X~ Y residue preceding the cleavage site is 59-4ml (with three side-chains of molal volume >60 ml), the two imino acids following the potential cleavage site are in neighboring triplets, and six charged residues are found in the overall region. Of the six charged residues, the two Hyl residues (in subsites P5 and P'~,) are glycosylated (Babel & Glanville, 1984), increasing the average side-chain molal volume preceding the cleavage site. (25) Mouse a l(IV) GlyloTo-Leu: There are only 17% imino acids preceding the potential cleavage site, the average side-chain molal volume per X~ Y residue preceding the cleavage site is 54.7 ml (with three side-chains of molal volume

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>60 ml), the two imino acids following the potential cleavage site are in neighboring triplets, and six charged residues are found in the overall region. Of the six charged residues, the two Hyl residues (in subsites P5 and P'~) are probably glycosylated (Schuppan et al., 1982). (26) Bovine a I(IV) Gly~07o-Leu: There are only 8-3% imino acids preceding the potential cleavage site, the average side-chain molal volume per X / Y residue preceding the cleavage site is 47.9 ml (with two side-chains of molal volume >60 ml), there are 25% imino acids following the cleavage site, and four charged residues are found in the overall region. One residue (subsite P~) has not been identified. (27-29) Human, Mouse and Bovine al(IV) Glyl~95-Leu: There are only 8.3% imino acids preceding the potential cleavage site, the average side-chain molal volume per X / Y residue preceding the cleavage site is 52.7-54.8 ml (with two side-chains of molal volume >60 ml), the two imino acids following the potential cleavage site are in neighboring triplets, Hyp is found in subsite P2, and five charged residues are found in the overall region. Of the five charged residues, the three Hyl residues (in subsites P~, Ps, and P'~j) are glycosylated (Babel & Glanville, 1984; Schuppan et al., 1984), significantly increasing the average side-chain molal volume preceding the cleavage site. (30) Human a2(IV) Gly1389-Ile: There are only 25% imino acids preceding the potential cleavage site, Pro is found in subsite P,_, a charged residue is found following the potential cleavage site (Lys in subsite P~), and the G I y - X - Y repeating sequence is not present in subsites P'6-P'~2. This is a gene-derived sequence, so it is not known how many, if any, of the Pro and Lys residues are hydroxylated and/or glycosylated. (31) Human ct5(IV) Gly54~-Leu: The average side-chain molal volume per X~ Y residue preceding the cleavage site is 51.8 ml (with three side-chains of molal volume >60 ml), Pro is found in subsite P_,, two charged residues are found following the potential cleavage site, and the G l y - X - Y repeating sequence is interrupted in subsites P~-P~. This is a gene-derived sequence, so it is not known how many, if any, of the Pro and Lys residues are hydroxylated and/or glycosylated; thus, the calculated average side-chain molal volume represents a minimum value. Analysis of all Gly-Ile-Ala, Gly-Leu-Ala, Gly-Ile-Leu, and Gly-Leu-Leu sequences clearly shows that non-cleaved regions contain several features that differ significantly from the cleavage site model. It needs to be ascertained, however, if triple-helical collagen structure can indeed by altered by the criteria defined in the model such that enzyme binding and/or hydrolysis is affected. One of the implications of the cleavage site model is that the region of high imino acid content distinguishes itself from the rest of the molecule by being more tightly "wound", and that a small change in imino acid content could "'unwind" this region slightly. Theoretical calculations for the lowest free energy conformations of the collagen models (Gly-Pro-Pro)4, (Gly-Pro-Hyp)4, (Gly-Pro-Ala)4, and (Gly-Ala-Pro)4 support this hypothesis. Conformational energy calculations for (Gly-Pro-Pro)4 and (Gly-Pro-Hyp)4 have shown that collagen-like triple-helical coiled-coil structures with screw symmetry are the most favored, i.e. have the lowest potential energies (-112.8 and -100-0kcal mol -~, respectively) (Miller & Scheraga, 1976;

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597

Miller et al., 1980a). For (Gly-Pro-Ala)4, the potential energy of the collagen-like structure is higher (-63.1kcal mol-l), with several alternative triple-helical and single-stranded structures of comparable energies (Miller et ai., 1980b). The collagenlike structure for (Gly-Ala-Pro)4 has an even higher potential energy (-31.0 kcalmo1-1) (Nemethy et al., 1980). The lowest potential energy conformation for (GlyAla-Pro)4 is not a collagen-like triple-helix, but rather many alternative structures of different symmetries (Nemethy et al., 1980). Thus, the absence of an imino acid in either the X or Y position can alter the lowest potential energy conformation, enabling the molecule to have local regions of decreased triple-helicity. Due to neighbor-neighbor interactions, replacement of an imino acid by an amino acid in the X position results in more alternative low energy conformations than a similar replacement in the Y position (Nemethy, 1981). A second implication of the cleavage site model is that the region of low imino acid content distinguishes itself from the rest of the molecule by micro-unfolding or loop formation. Micro-unfolding within the collagen triple-helix has been suggested based on decreased thermal stability of single amino acid-mutated otl(I) chains (Westerhausen et al., 1990) and conformational analysis of a peptide model of the a l ( I ) chain (Scaria et al., 1990). Conformational energy calculations have examined whether loop formation could be favored within a triple-helix (Mattice et al., 1988; Nemethy & Scheraga, 1989). The free energy contributions in going from a triple-helix to a loop structure are (i) the energy of breaking non-covalent interactions between the three strands in the triple-helix (Miller & Scheraga, 1976), (ii) the change of conformational energy of the individual strands (Miller & Scheraga, 1976), (iii) the change in the free energy of hydration of the strands (Nemethy & Scheraga, 1989), and (iv) the conformational entropy of formation of the loop (Mattice et al., 1988). Loop formation was energetically probable for 20-30 residue triple-helical regions containing no or low imino acid content (Mattice et al., 1988; Nemethy & Scheraga, 1979). These results collectively suggest that the low imino acid region following the mammalian collagenase cleavage site could unfold and form a loop. A third implication of the cleavage site model is that a low charged residue density is favorable for collagenase binding and hydrolysis. This is most probably due to the significant effect charged residues have on collagen structure (Nemethy & Scheraga, 1982). Charged residues Asp, Glu, Lys, and Arg located in either the X or Y position in collagen triplets can form strong intra- and interstrand hydrogen bonds (Nemethy & Scheraga, 1982). In some cases, such as Asp in X or Y, hydrogen bonding results in conformations that are of lower potential energy than the previously discussed "collagen-like" conformation (Nemethy & Scheraga, 1982). Thus, charged residues in the region preceding the collagenase cleavage site (where the "tightly triple-helical" conformation must be strictly maintained) or following the cleavage site (where the "loop" conformation is required) will be detrimental for enzyme binding and/or hydrolysis. The mamrhalian collagenase cleavage site model proposed here is consistent wth all experimental and theoretical results on collagen catabolism and structure. It also explains well why many collagen sequences containing potentially cleavable bonds

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are not cleaved. The recent work of Wu et al. (1990) on the mammalian collagenase cleavage of mutant collagens strongly supports this model. For example, the cleavage site model proposes that a 25 amino acid region dictates mammalian collagenase behavior. Based on peptide sequence specificity work, the active site of mammalian collagenases was shown to extent to at least eight residues of the substrate, i.e. subsites P4-P~ (Fields et al., 1987; Fields, 1988; Netzel-Arnett et al., 1991). When the two Val residues in subsites P~ and P~ of the a 1(I) chain of collagen are mutated (by site-directed mutagenesis) to two Ala residues, the relative rate of mammalian collagenase cleavage is slowed (Wu et al., 1990). Thus, the number of substrate residues that effects collagenase cleavage is at least 12, i.e. subsites P4-P~. Also, collagenase cleavage was abolished by the double mutation of Pro for Gin in subsite P2 and Pro for Ala in subsite P~ (Wu et al., 1990). As stated in the cleavage site model, an imino acid in subsite P2 and the presence of imino acids in the P' subsites of the potential cleavage site are detrimental for collagenase binding and/or hydrolysis. This result for mammalian collagenase hydrolysis of mutant collagen not only supports the cleavage site model, but also demonstrates that only a few of the criteria of the model need be altered for a sequence to become resistant to collagenase hydrolysis. In addition, two non-cleavable mutant a 1(I) chains will prevent hydrolysis of a viable t~2(I) chain in triple-helical type I collagen (Wu et al., 1990). Although the model explains well the seven non-cleaved a2(I) chains shown in Table 2, these chains may not be cleaved simply because they are surrounded by two non-cleavable oil(I) chains. The unique features of the mammalian collagenase cleavage site model may also be important for other protein interactions with interstitial collagens. For example, the collagen cell attachment protein from fibroblasts and Chinese hamster ovary cells binds rat skin type I collagen exclusively between residues 757-791, with cell binding destroyed by cleavage of the 775-776 bond, and rat type II collagen exclusively between residues 552-822 (Kleinman et al., 1976, 1978). Since the model developed here incorporates residues 763-787 of types I, II, and III collagens, some of the criteria may prove insightful for understanding mechanisms of cell binding to interstitial collagens. I wish to thank Drs Stephen Krane, Jerome Gross, and Henning Birkedal-Hansen for helpful comments, insights, and suggestions prior to the preparation of this manuscript, Dr Harald Tschesche for the reprints and preprints regarding his laboratory's results with human polymorphonuclear leukocyte procollagenase, and Cynthia G. Fields for constructing Fig. 1. This work was partially supported by the 1989 Matrix Metalioproteinase Conference via a Young Investigator Award to G.B.F. REFERENCES BABEL, W. & GLANVILLE, R. W. (1984). Structure of human-basement-membrane (type IV) collagen: complete amino acid sequence of a 914-residue-long pepsin fragment from the a 1(IV) chain. Eur. J. Biochem. 143, 545-556. BALLAN, G., CLECK, E. M., HERMODSON, M. A. & BORNSTEIN, P. (1972). Structure of rat skin collagen aI-CB8: amino acid sequence of the hydroxylamine-produced fragment HA2.. Biochemistry 11, 3798-3806.

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BEAR, R. S. (1952). The structure of collagen fibrils. Adv. Protein Chem. VII, 69-160. BHATNAGAR, R. S., PA'F'FABIRAMAN) N., SORENSEN, g. R., LANGRIDGE, R., MACELROY, R. D. R, RENUGOPALAKRISHNAN, V. (1988). Inter-chain proline:proline contacts contribute to the stability of the triple helical conformation. J. Biomol. Struct. Dynam. 6, 223-233. BIRKEDAL-HANSEN, H., BIRKEDAL-HANSEN, B., WINDSOR, L. J., LIN, H.-Y., TAYLOR, R. E. & MOORE, W. G. I. (1989). Use of inhibitory (anti-catalytic) antibodies to study extracellular proteolysis. lmmun. Invest. 18, 211-224. BIRKEDAL-HANSEN, H., TAYLOR, R. E., BHOWN, A. S., KATZ, J., LIN, H.-Y. & WELLS, B. R. (1985). Cleavage of bovine skin type Ill collagen by proteolytic enzymes: relative resistance of the fibrillar form. J. biol. Chem. 260, 16411-16417. BORNSTEIN, P. & TRAUB, W. (1979). The chemistry and biology of collagen. In: The Proteins Voi. 4 3rd Ed. (Neurath, H. & Hill, R. L., eds) pp. 411-631. Orlando, FL: Academic Press. BROWN, R. A., HUKINS, D. W. L., WEISS, J. B. & TWOSE, T. M. (1977). Do mammalian collagenases and DNA restriction endonucleases share a similar mechanism for cleavage site recognition? Biochem. biophys. Res. Commun. 74, 1102-1108. BURGE, R. E. & HYNES, R. D. (1959). The thermal denaturation of collagen in solution and its structural implications. 3. molec. Biol. 1, 155-164. DEWES, H., F1ETZEK, P. P. & KUHN, K. (1979). The covalent structure of calf skin type III collagen I I: the amino acid sequence of the cyanogen bromide peptide ct 1( I I I )C B 1,8,10,2 ( positions 223-402). Hoppe-Seylers Z. Physiol. Chem. 360, 821-832. DIXlT, S. M., MAINARDI, C. L., SEYER, J. M. & gANG, A. H. (1979). Covalent structure of collagen: amino acid sequence of a2-CB5 of chick skin collagen containing the animal collagenase cleavage site. Biochemistry 18, 5416-5422. DIXIT, S. M., SEYER, J. M. & gANG, A. H. (1977). Covalent structure of collagen: isolation of chymotryptic peptides and amino acid sequence of the amino-terminal region of a2-CB3 from chick skin. Eur. J. Biochem. 73, 213-221. DOLZ, R. & HEIDEMANN, E. (1986). Influence of different tripeptides on the stability of the collagen triple helix I: analysis of the collagen sequence and identification of typical tripeptides. Biopolymers 25, 1069-1080. FIELDS, G. B. (1988). The application o f solid phase peptide synthesis to the study of structure-function relationships in the collagen-collagenase system. Ph.D. Thesis, Florida State University, Tallahassee, FL. FIELDS, G. B., O'VI'ESON, K. M., FIELDS, C. G. &. NOBLE, R. L. (1990a). The versatility of solid-phase peptide synthesis. In: Innovation and Perspectives in Solid-Phase Synthesis (Epton, R., ed.) pp. 241-260. Birmingham, U.K.: Solid Phase Conference Coordination, Ltd. FIELDS, G. B., NETZEL-ARNETT, S. J., WINDSOR, L. J., ENGLER, J. A., B1RKEDAL-HANSEN, H. & VAN WART, H. E. (1990b). Proteolytic activities of human fibroblast collagenase: hydrolysis of a broad range of substrates at a single active site. Biochemistry 29, 6670-6677. FIELDS, G. B. & VAN WART, H. E. (1991). Unique features of the tissue collagenase cleavage site in interstitial collagens. Matrix: Coll. Rel. Res. (Suppl.), in press. FIELDS, G. B., VAN WART, H. E. & BIRKEDAL-HANSEN, H. (1987). Sequence specificity of human skin fibroblast collagenase: evidence for the role of collagen structure in determining the collagenase cleavage site. 3. biol. Chem. 262, 6221-6226. FIN1, M. E., PLUCINKA, I. M., MAYER, A. S., GROSS, R. H. & BRINCKERHOFF, C. F. (1987). A gene for rabbit synovial cell collagenase: member of a family of metalloproteinases that degrade the connective tissue matrix. Biochemistry 26, 6156-6165. FRANCIS, G., BUTLER, W. T. &- FINCH, J. E. F. JR. (1978). The covalent structure of cartilage collagen: amino acid sequence of residues 552-661 of bovine a l(II) chains. Biochem. 3. 175, 921-930. GERMANN, H.-P. & HEIDEMANN, E. (1988). A synthetic model of collagen: an experimental investigation of the triple-helix stability. Biopolymers 27, 157-163. GLANVILLE, R. W., BREITKREUTZ, D., MEITINGER, M. ,f" FIETZEK, P. P. (1983). Completion of the amino acid sequence of the a 1 chain from type I calf skin collagen: amino acid sequence of a I(I)BH. Biochem. J. 215, 183-189. GOLDBERG, G. I., WILHELM, S. M., KRONBERGER, A., BAUER, E. A., GRANT, G. A. & EISEN, A. Z. (1986). Human fibroblast collagenase: complete primary structure and homology to an oncogene transformation-induced rat protein. 3. biol. Chem. 261, 6600-6605. GOLf, U. B., FIELDS, G. B. & VAN WART, H. E. (1991). Synthetic triple-helical models for the collagenase cleavage site 'in interstitial collagens. Matrix: Coll. Rel. Res. (Suppl.), in press. GROSS, J., HARPER, E., HARRIS, E. D. JR., McCROSKEY, P. A., HIGHBERBER, J. H., CORBET1r, C. & gANG) A. H. (1974). Animal collagenases: specificity of action, and structures of the substrate cleavage site. Biochem. biophys. Res. Commun. 61,605-612.

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GROSS, J., HIGHBERGER, J. H., JOH NSON-WINT, B. & BISWAS, C. (1980). Mode of action and regulation of tissue collagenases. In: Collagenase in Normal and Pathological Connective Tissues (Woolley, D. E. & Evanson, J. M., eds) pp. 11-35. New York: John Wiley & Sons. HASTY, K. A., POU RMOTABBED, Z. F., GOLDBERG, G. I., THOMPSON, J. P., SPl NELLA, D. G., STEVENS, R. M. & MAINARDI, C. L. (1990). Human neutrophil collagenase: a distinct gene product with homology to other matrix metalloproteinases. J. biol. Chem. 265, 11421-11424. HIGHBERGER, J. J., CORBETT, C., DIXIT, S. N., YU, W., SEYER, J. M., KANG, A. M. & GROSS, J. (1982). Amino acid sequence of chick skin collagen a I(I)-CB8 and the complete primary structure of the helical portion of the chick skin collagen al(1) chain. Biochemistry 21, 2048-2055. HIGHBERGER, J. J., CORBETT, C. & GROSS, J. (1979). Isolation and characterization of a peptide containing the site of cleavage of chick skin collagen by animal collagenases. Biochem. biophys. Res. Commun. 89, 202-208. HOFMANN, H., F1ETZEK, P. P. & KUHN, K. (1978). The role of polar and hydrophobic interactions for the molecular packing of type I collagen. J. molec. Biol. 125, 137-165. HOSTIKKA, S. L. & TRYGGVASON, K. (1988). The complete primary structure of the a2 chain of human type IV collagen and comparison with the a l ( I V ) chain. J. biol. Chem. 263, 19488-19493. INOUYE, K., KOBAYASHI, Y., KYOGOKU, Y., KISHIDA, Y., SAKAKIBARA, S. & PROCKOP, D. J. (1982). Synthesis and physical properties of (hydroxyproline-proline-glycine)t0: hydroxyproline in the Xposition decreases the melting temperature of the collagen triple helix. Arch. Biochem. Biophys. 219, 198-203. INOUYE, K., SAKAKIBARA, S. & PROCKOP, D. J. (1976). Effects of the stereo-configuration of the hydroxyl group in 4-hydroxyproline on the triple-helical structures formed by homogeneous peptides resembling collagen. Biochem. Biophys. Acta 420, 133-141. IUPAC-IUB Commission of Biochemical Nomenclature (1972). J. biol. Chem. 247, 977-983. KLEINMAN, H. K., McGooDWIN, E. B. & KLEBE, R. J. (1976). Localization of the cell attachment region in types I and I1 collagen. Biochem. biophys. Res. Commun. 72, 426-432. KLE1NMAN, H. K., McGoODWlN, E. B., MARTIN, G. R., KLEBE, R. J., FIETZEK, P. P. & WOOLLEY, D. E. (1978). Localization of the binding site for cell attachment in the col(I) chain of collagen. J. biol. Chem. 253, 5642-5646. KNAOPER, V., KR.~MER, S., REINKE, H. & TSCHESCHE, H. (1990a). Characterization and activation of procollagenase from human polymorphonuclear leucocytes. Eur. J. Biochem. 189, 295-300. KN.~UPER, V., KR.~MMER, S., REINKE, H. & TSCHESCHE, H. (1990B). Partial amino acid sequence of human polymorphonuclear leukocyte procollagenase. Biol. Chem. Hoppe-Seyler 371 (Suppl.), 295-304. KN.~UPER, V., KR.AMER, S., REINKE, H. & TSCHESCHE, H. 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