Several peptides containing intermolecular crosslinks were isolated from tryptic digests of the insoluble matrix of human leiomyoma and calf aorta. From leiomyoma type 111 collagen, two crosslinked peptides were isolated. The peptide [T,(III)], is a dimer which contains the 73 amino-terminal residues of two, zero-D staggered, crosslinked aI(II1) chains (D being the distance by which molecules or chains are staggered relative to each other). After deblocking with pyroglutamate aminopeptidase, its amino-terminal amino acid sequence was determined to be < Glu-Tyr-Asp-Ser-Tyr-Asp-Val-Xaa-Ser-Gly-Val-Ala-Val-Gly-Gly-Leu-Ala-Gly-Tyr-Hyp-Gly-. The involvement of [TN(II1)l2 in the formation of 4D-staggered intermolecular bonds was demonstrated by sequence analysis of [T,(ITI)], x T(II1). For this three-chained peptide, a double sequence which corresponded to T,(III) and T(III), a nine-residue peptide from the carboxy-terminal helical crosslinking site of type I11 collagen, was found.Crosslinks between type I and type I11 collagens were identified by isolating a peptide T,(III) x T,(I) from leiomyoma. This peptide was characterized, first by digesting it with collagenase giving rise to Col,(III) x Col,(I) and then chymotrypsin giving C(II1) and C(1). C(1) and C(II1) are peptides characteristic for type I and type 111 collagens, respectively. Further, three-component crosslink-containing peptides, from both type I and type I11 collagen, were isolated from aorta and leiomyoma. The peptides Col,(III) x Col,(I) x T(II1) from leiomyoma and Col,(III) x Col,(I) x T(1) from aorta are crosslinked amino-terminal regions of OD-staggered type I and type I11 molecules, joined either to an adjacent 4D-staggered type 111 or type I molecule. Crosslinked peptides involving only type I11 collagen molecules were also isolated from calf aorta [colN(III)]2 and [CoI,(III)], x T(III)].As intramolecular bonds between type 1 and 111 collagens can be excluded the isolation of these crosslinked peptides demonstrated that type I and type I11 collagen molecules are present within the same collagen fibril with OD stagger, bound by intermolecular crosslinks. The significance of these findings with respect to the proposed models for collagen fibres and the physiological properties of the extracellular matrix, are discussed.Covalent crosslinking of collagen molecules within a fibre, which is the final step in the formation of a collagen matrix, has been described for type I, type IT and type I11 collagens (for recent review see [l]). Structural investigations on these mol- Nomenclature of' Crosslinked Peptides. Due to the absence of a systematic nomenclature for crosslinked peptides derived from insoluble collagens, the following system was developed. The type of collagen is denoted by roman numerals in brackets. The enzyme cleavage which produced the peptides is denoted by Col, T or C for bacterial collagenase, trypsin or chymotrypsin, respectively. The subscript N indicates that the peptide contains the amino-terminal nonhelical region of the collagen...

Solid-phase synthesis of triple-helical peptides, including native collagen III sequences, was started with a trimeric branch, based upon the lysine dipeptide [Fields, C. G., Mickelson, D. J., Drake, S. L., McCarthy, J. B., and Fields, G. B. (1993) J. Biol. Chem. 268, 14153-14160]. Branch synthesis was modified, using TentaGel R as resin, p-hydroxybenzyl alcohol (HMP) as linker, Dde as N(epsilon)-protective group, and HATU/HOAT as coupling reagent. Three homotrimeric sequences, each containing the Gly 606-Gly 618 portion of human type III collagen, were added to the amino functions of the branch. The final incorporation of GlyProHyp triplets, stabilizing the collagen III triple helix, was performed by using protected GlyProHyp tripeptides, each containing tert-butylated hydroxyproline [P(tBu)] instead of hydroxyproline (P). Among the protected tripeptides FmocP(tBu)PG, FmocPP(tBu)G, and FmocGPP(tBu), prepared manually on a chlorotrityl resin, incorporation of FmocPP(tBu)Gly was best suited for synthesis of large and stable peptides, such as PPG(8), containing 8 (PPG)(3) trimers (115 residues, 10 610 Da). The structures of five peptides, differing from each other by the type and number of the triplets incorporated, were verified by MALDI-TOF-MS. Their conformations and thermodynamic data were studied by circular dichroism and differential scanning calorimetry. Except for PPG(8), containing 8 (PPG)(3) trimers with hydroxyproline in the X position and adopting a polyproline II structure, all peptides were triple-helical in 0.1 M acetic acid and their thermal stabilities ranged from t(1/2) = 39. 4 to t(1/2) = 62.5 degrees C, depending on the identity and number of the triplets used. Similar values of the van't Hoff enthalpy, DeltaH(vH), derived from melting curves, and the calorimetric enthalpy, DeltaH(cal), obtained from heat capacity curves, indicate a cooperative ratio of CR = DeltaH(vH)/DeltaH(cal) = 1, establishing a two-state process for unfolding of THP(III) peptides. The independence of the transition temperatures t(1/2) on peptide concentration as well as equilibrium centrifugation data indicate monomolecular dimer(f) to dimer(u) (F(2) <--> U(2)) transitions and, in addition, bimolecular dimer(f) to monomer(u) transitions (F(2) <--> 2U). The dominance of the concentration-independent monomolecular reaction over the concentration-dependent bimolecular reaction makes thermal unfolding of THP(III) peptides appear to be monomolecular. If one designates the molecularity described by the term pseudomonomolecular, unfolding of the dimeric peptides PPG(6-8) follows a two-state, pseudomonomolecular reaction.

The network of the fibrillar collagens I, III, and V, extracted from fetal calf skin and cleaved with cyanogen bromide, was studied by means of ultraviolet matrix-assisted laser desorption ionization time-of-flight mass spectrometry (UV-MALDI MS). Nearly all of the expected cyanogen bromide peptides of the different alpha chains were detected. Distinct peptides are identified that can serve as a reference signal for the individual alpha-chains. Homo- and heterotypic cross-linking patterns, some of which have not been described before for bovine collagen, are indicated by comparison of the mass spectrometric data with documented amino acid sequences. Potential cross-linking mechanisms are discussed. For example, the mass spectrometric data suggest that the formation of heterotypic I/III and I/V fibrils is substantially determined by the telo-regions of type I collagen, which are covalently connected to the corresponding helical and nonhelical cross-linking domains of adjacent molecules either by 4D or 0D-stagger bonds. The chemical nature of the cross-links can be concluded. The data also indicate a disturbed formation of heterotypic fibrils. Finally, collagen glycosylation can also be identified.

Fibril-forming collagen proteins of the types I, III, and V were extracted from fetal calf skin, purified by differential salt precipitation, and analyzed by infrared matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (IR-MALDI-TOF-MS). Glycerol was used as liquid IR-MALDI matrix. Noncovalently bound triple helices of the types I and V were detected from the NaCl precipitate. After heating at 43 degrees C for 10 min, resulting in the dissociation of the triple helix, the single alpha-chain subunits were detected. For type I, mass spectra acquired from molecular sieve chromatography fractions revealed the presence of further substructures of dimeric type and of supramolecular complexes up to the tetramer. Triple helices of type III, stabilized by covalent disulfide bonds, were detected from the total protein precipitate also after heat treatment. For type III, even hexamer and nonamer structures with molecular weights close to 600 and 900 kDa were recorded. For comparison, ultraviolet (UV-)MALDI-MS measurements with 2,5-dihydroxybenzoic acid as matrix were carried out with some of the samples. Here, only the single alpha-chains were detected with significantly reduced sensitivity.