Identification and Structural Characterization of Human Collagenase3

The identification of human collagenase-3 resulted from studies intended to identify the presence of new proteolytic enzymes in breast carcinomas.1 One of the distinctive features of malignant tumors is their ability to invade normal tissues and spread to distant sites, in the process giving rise to metastasis.2 These events involve degradation of the different macromolecular components of the extracellular matrix, and require the combined action of several proteolytic systems. Among various enzymes implicated in tumor invasion and metastasis, a number of previous studies focused on matrix metalloproteinases because of their unique ability to collectively degrade all connective tissue protein components at neutral pH.3-5

These considerations, led us to speculate that samples of human tumor specimens were an appropriate starting material to identify putative novel members of the MMP family potentially involved in the spread of cancer. To test this idea, we designed a PCR-based homology cloning strategy, using RNA isolated from breast carcinomas and degenerate oligonucleotides encoding structural motifs conserved in MMPs. When we initiated this work, a total of nine human MMPs had been isolated and characterized at the amino acid sequence level (interstitial collagenase, neutrophil collagenase, gelatinases A and B, stromelysins-1, -2, -3, matrilysin, and macrophage metalloelastase).3,4 A comparison of their amino acid sequences revealed two sequences conserved in all of them, the activation locus (PRCGVPD) and the Zn-binding site (VAAHEXGH). After synthesizing two degenerate oligonucleotides encoding these conserved motifs and performing RT-PCR of total RNA isolated from a mammary carcinoma, a band of the expected size was obtained and cloned. Analysis of the nucleotide sequence of different clones revealed that some of them had a sequence similar to, but distinct from all previously characterized human MMPs. Screening of a breast cancer cDNA library prepared from the same tumor employed for the RT-PCR experiment and using the PCR-generated fragment as probe, we identified a positive clone with an insert of 2.7 kb. The isolated cDNA encoded a polypeptide of 471 amino acids,

Collagenases, edited by Warren Hoeffler. ©1999 R.G. Landes Company.

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human

M-HPGVLAAF

LFLSWTHCRA

LPLP-SGGDE

DDLSEEDLQF

AERYLRSYYH

P-TNLAGILK

ENAASSMTER

LREMQSFFGL

EVTGKLDDNT

mouse

M-HSAILATF

FLLSWTPCWS

LPLPYGDDDD

DDLSEEDLVF

AEHYLKSYYH

P-ATLAGILK

KSTVTSTVDR

LREMQSFFGL

EVTGKLDDPT

rat

M-HSAILATF

FLLSWTHCWS

LPLPYGDDDD

DDLSEEDLEF

AEHYLKSYYH

P-VTLAGILK

KSTVTSTVDR

LREMQSFFGL

DVTGKLDDPT

Xenopus

MAPSSLSVFV

LSLSFTYC—

LSAPVSQDED

SELTPGALQL

AEHYLNRLYS

SSSNPAGMLR

MKDVNSVETK

LREMQSFFGL

EVTGKLNEDT

newt

MMPSVLSAAI

FFLSLAFG—

LPVPVPHERD

SDVTEQELRL

AEKYLKTFYV

A-SDHAGIMT

KKGGNALASK

LREMQSFFDL

EVTGKLDEDT

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********

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human

LDVMKKPRCG

VPDVGEYNVF

PRTLKWSKMN

LTYRIVNYTP

DMTHSEVEKA

FKKAFKVWSD

VTPLNFTRLH

DGIADIMISF

GIKEHGDFYP

mouse

LDIMRKPRCG

VPDVGEYNVF

PRTLKWSQTN

LTYRIVNYTP

DMSHSEVEKA

FRKAFKVWSD

VTPLNFTRIY

DGTADIMISF

GTKEHGDFYP

rat

LDIMRKPRCG

VPDVGVYNVF

PRTLKWSQTN

LTYRIVNYTP

DISHSEVEKA

FRKAFKVWSD

VTPLNFTRIH

DGTADIMISF

GTKEHGDFYP

Xenopus

LDIMKQPRCG

VPDVGQYNFF

PRKLKWPRNN

LTYRIVNYTP

DLSTSDVDRA

IKKALKVWSD

VTPLNFTRLR

TGTADIMVAF

GKKEHGDYYP

newt

LEVMKQPRCG

VPDVGEYNVF

PRSLKWPRFN

LTYRIENYTP

DMTHAEVDRA

IKKAFRVWSE

VTPLNFTRLR

SGTADIMISF

GTKEHGDFYP

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*****

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****** ***

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human

FDGPSGLLAH

AFPPGPNYGG

DAHFDDDETW

TSSSKGYNLF

LVAAHEFGHS

LGLDHSKDPG

ALMFPIYTYT

GKSHFMLPDD

DVQGIQSLYG

mouse

FDGPSGLLAH

AFPPGPNYGG

DAHFDDDETW

TSSSKGYNLF

IVAAHELGHS

LGLDHSKDPG

ALMFPIYTYT

GKSHFMLPDD

DVQGIQFLYG

rat

FDGPSGLLAH

AFPPGPNLGG

DAHFDDDETW

TSSSKGYNLF

IVAAHELGHS

LGLDHSKDPG

ALMFPIYTYT

GKSHFMLPDD

DVQGIQSLYG

Xenopus

FDGPDGLLAH

AFPPGEKIGG

DTHFDDDEMF

STDNKGYNLF

VVAAHEFGHA

LGLDHSRDPG

SLMFPVYTYT

ETSRFVLPDD

DVQGIQALYG

newt

FDGPNGLLAH

AFPPGQRIGG

DTHFDDDETF

TSGSNGYNLF

IVAAHEFGHA

LGLDHSRDPG

SLMYPVYSYT

EPSRFLLPDD

DVQGIQSLYG

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human

PGDEDPNPKH

PKTPDKCDPS

LSLDAITSLR

GETMIFKDRF

FWRLHPQQVD

AELFLTKSFW

PELPNRIDAA

YEHPSHDLIF

IFRGRKFWAL

mouse

PGDEDPNPKH

PKTPEKCDPA

LSLDAITSLR

GETMIFKDRF

FWRLHPQQVE

AELFLTKSFW

PELPNHVDAA

YEHPSRDLMF

IFRGRKFWAL

rat

PGDEDPNPKH

PKTPEKCDPA

LSLDAITSLR

GETMIFKDRF

FWRLHPQQVE

PELFLTKSFW

PELPNHVDAA

YEHPSRDLMF

IFRGRKFWAL

Xenopus

SGNRDPHPKH

PKTPEKCDPD

LTIDAITELR

GEKMIFKDRF

FWRVHPQMTD

AELVLIKSFW

PELPNKIDAA

YEHPAKDLIY

IFRGKKFWAL

newt

PGNRDPNPKH

PKTPEKCDPE

LSLDAITEMR

GEKLIFKDRF

FWRQHPQMTD

VELVLIRNFW

PELPSKIDAA

YEYPEKDLIY

IFRGRKFWAL

^ -k -k -k *

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human

NGYDILEGYP

KKISELGLPK

EVKKISAAVH

FEDTGKTLLF

SGNQVWRYDD

TNHIMDKDYP

RLIEEDFPGI

GDKVDAVYEK

NGYIYFFNGP

mouse

NGYDILEGYP

RKISDLGFPK

EVKRLSAAVH

FENTGKTLFF

SENHVWSYDD

VNQTMDKDYP

RLIEEEFPGI

GNKVDAVYEK

NGYIYFFNGP

rat

NGYDIMEGYP

RKISDLGFPK

EVKRLSAAVH

FEDTGKTLFF

SGNHVWSYDD

ANQTMDKDYP

RLIEEEFPGI

GDKVDAVYEK

NGYIYFFNGP

Xenopus

NGYDFVEDYP

KKLHELGFPK

TLKAIDAAVY

NKAIGKTLFF

AEDSYWSFDE

EARTMDKGFP

RLISEDFPGI

GEKVDAAYQR

NGYIYFFNGA

newt

NGYDILADYP

KKIQELGFPK

SLRTIDAAVY

NRAMGKTLFF

TGEKYWSFDE

EKQTVEKGYP

RFIADDFPGI

GETVDAAYQR

NGYIYFFSGS

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human

IQFEYSIWSN

RIVRVMPANS

ILWC

mouse

IQFEYSIWSN

RIVRVMPTNS

ILWC

rat

IQFEYSIWSN

RIVRVMPTNS

LLWC

Xenopus

LQFEYSIWSK

RITRILKTNF

VLMC

newt

LQFEYSTWSN

KVIRVLKTNS

ILWC

Fig. 3.1. Comparison of the amino acid sequences of collagenase-3 (MMP-13) from different species. The amino acid sequences of human, mouse, rat, Xenopus, and newt collagenase-3 were extracted from the SwissProt data base and the multiple alignment was performed with the PILEUP program of the University of Wisconsin Genetics Computer Group package. Identical residues in all five sequences are indicated by asterisks.

having all the structural features characteristic of MMPs, including the predomain with a hydrophobic leader sequence, the pro-domain having the PRCGVPD activation locus, the Zn-binding site of the catalytic domain, and a C-terminal extension with sequence similarity to hemopexin (Fig. 3.1). This novel human MMP contained in its amino acid sequence several residues specific to the collagenase subfamily of MMPs (Tyr-214, Asp-235, and Gly-237), but lacked the 9-residue insertion present in the stromelysins and in the fibronectin domain characteristic of the gelatinases. According to these structural characteristics, this human MMP was designated collagenase-3 (MMP-13), since it represented the third member of the subfamily composed at that time of fibroblast and neutrophil collagenases.1

Pairwise comparisons for amino acid sequence similarities between collagenase-3 and other human MMPs confirmed that the highest percentage of identities (about 50%) was found with the human collagenases. However, and somewhat surprisingly, when the sequence comparison was extended to all sequences present in the databank, a higher degree of identities was found with rat and mouse interstitial collagenases.6,7 Since their description, it had been assumed that these enzymes were the murine counterparts of human interstitial collagenase (MMP-1). However, the finding that they were more closely related to human collagenase-3 indicated that these murine enzymes represented counterparts of MMP-13 instead of MMP-1. To date, there is no evidence for a homologous MMP-1 in either rat or mouse, and the possibility exists that rodents do not possess this gene.

To avoid future confusion with the nomenclature of the collagenase subfamily of MMPs, I would like to propose the use of collagenase-1 to designate interstitial or fibroblast collagenase (MMP-1), and collagenase-2 for neutrophil collagenase (MMP-8). In fact, the novel human collagenase is also produced by fibroblast and inflammatory cells,8-10 whereas neu-trophil collagenase is also synthesized by articular chondrocytes,11 also a source of collage-nase-3.12-15 Consequently, cellular origin and tissue distribution are not distinctive characteristics of these enzymes. Furthermore, this nomenclature system would facilitate the description of as yet uncharacterized human collagenases, as may be the case of the putative human homologue of the recently described collagenase-4 (MMP-18) identified in Xenopus laevis.16

The collagenase-3 family has recently grown with the finding of homologue enzymes in two additional species: Xenopus laevis and Cynopus pyrrhogaster (newt). The Xenopus collagenase-3 was identified as a thyroid hormone-induced gene during tail resorption,17 whereas the newt homologue was cloned from early bud-stage regenerating limbs.18 A comparison of the amino acid sequences of the five collagenase-3 sequences reported so far is shown in Figure 3.1. The percentage identities with the human enzyme ranges from 86% with the murine enzymes to about 60% with Xenopus collagenase-3. A more detailed analysis of these sequences reveals an uneven distribution of conserved sequences between them, especially between the amphibian enzymes and the human and murine sequences. The catalytic domains are highly conserved and contain all the essential residues for catalytic properties as well as the distinctive residues of the collagenase subfamily. In contrast, the signal sequence, the prodomain and hemopexin domains show a higher divergence between species. The availability of all five sequences will be very useful for future studies directed at identifying specific residues responsible for the distinctive enzymatic properties of collage-nase-3.

In research that will aid in the structural analysis of collagenase-3, recent studies have solved the crystal structure of the C-terminal hemopexin-like domain of the human enzyme by molecular replacement.19 This structure reveals a disk-like shape with the polypeptide chain folded into a p-propeller structure of pseudo 4-fold symmetry. The four propeller blades are arranged around a funnel-shaped tunnel that harbors two calcium and two chloride ions. This structure thus has a high degree of homology to the equivalent domain of porcine collagenase-1.20 The elucidation of the complete three-dimensional structure of collagenase-3 has been hampered by the high instability of the enzyme. In fact, collagenase-3 possesses a potent autoproteolytic activity, which leads to an extensive autolysis of the molecule, with the exception of the C-terminal domain, which remained intact.21 Nevertheless, the complete crystal structure of this enzyme must be elucidated to gain a better understanding of its functional relevance in both normal and pathological conditions.

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