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To establish the role of posttranslational modification in modulating the immune response to collagen, recombinant human type II collagen (rCII) was produced using a yeast expression system (rCIIpic) and a baculovirus expression system (rCIIbac). The biosynthesis of CII requires extensive posttranslational modification including the hydroxylation of prolyl and lysyl residues and glycosylation of selected hydroxylysyl residues. Amino acid analyses indicated that the rCIIbac was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived CII, whereas rCIIpic was adequately hydroxylated at prolyl residues but unhydroxylated at lysyl residues and had no glycosylation. When DBA/1 mice were immunized with rCII, rCIIpic induced a lower incidence of arthritis than tissue-derived CII, whereas rCIIbac induced an intermediate level of arthritis. The severity of the arthritis was significantly lower in mice immunized with rCIIpic compared with mice immunized with tissue-derived CII, whereas that of rCIIbac was intermediate. These data indicate that the degree of lysine hydroxylation and glycosylation plays a role in the induction of arthritis. The recombinant collagens were then compared with tissue-derived CII when given as i.v. or oral tolerogens to suppress arthritis. Both recombinant collagens were less potent than tissue-derived CII, and this decrease in arthritis was associated with a decrease in Ab response to CII. These data suggest that the degree of glysosylation affects the immune response to CII, so that underglycosylated CII is less effective in the induction of arthritis and in its ability to suppress collagen-induced arthritis.

Prolyl 4-hydroxylase (4-PH) catalyzes the formation of 4-hydroxyproline in -X-Pro-Gly- sequences and has a central role in the synthesis of all collagens. We report here on the cloning and characterization of the genes encoding the catalytic alpha(II) subunits of the human and mouse type II 4-PH [alpha(II)]2beta2 tetramers. The human and mouse genes are approximately 34.6 kb and 30.3 kb in size, respectively, and both consist of 16 exons. The translation initiation codons are located in exon 2, and the sizes of the exons consisting entirely of coding sequences are conserved in the two genes, varying from 54 to 240 bp, whereas the exons 1, containing the transcription initiation sites and 5' untranslated sequences, are 546 bp and 293 bp in the human and mouse, respectively. The sizes of the introns vary from 48 to 49 bp to over 8 kb in both genes. The 5' flanking regions contain no TATA box, but they and introns 1 contain several motifs that may act as transcription factor binding sites, including those for Sox9, which regulates chondrocyte-specific expression of collagens II, IX and XI. Unlike the human alpha(I) gene, the alpha(II) genes do not contain an alternatively spliced exon homologous to exon 9. However, a novel mutually exclusively spliced alternative exon 12a was identified in both genes. The nucleotide and amino-acid sequence identities between the 60-bp exon 12a and 66-bp exon 12b are about 35% and 45%, respectively, in both human and mouse genes. PCR analyses showed that both types of exon 12 are expressed in all tissues studied, except for adult leukocytes that expressed only mRNAs containing exon 12b sequences. Insect cell expression studies showed that a recombinant alpha(II) subunit containing amino acids coded by exon 12a associated with the beta subunit to form a fully active enzyme tetramer.

Four human genes, two of them encoding the proalpha1 and proalpha2 chains of type I procollagen and two of them the two types of subunit of prolyl 4-hydroxylase (4-PH), were integrated into the genome of Pichia pastoris. The proalpha1 and proalpha2 chains expressed formed type I procollagen molecules with the correct 2:1 chain ratio, and the 4-PH subunits formed an active enzyme tetramer that fully hydroxylated the proalpha chains. Chains lacking their N but not C propeptides formed pCcollagen molecules with the 2:1 chain ratio and, surprisingly, the expression levels of pCcollagen were 1.5-3-fold relative to those of procollagen. Both types of molecule could be converted by pepsin treatment to collagen molecules that formed native-type fibrils in vitro. The expression levels obtained for the pCcollagen using only single copies of each of the four genes and a 2 l fermenter ranged up to 0.5 g/l, indicating that it should be possible to optimize this system for high-level production of recombinant human type I collagen for numerous medical applications.

As type IX collagen is a minor cartilage component, it is difficult to purify sufficient amounts of it from tissues or cultured cells to study its structure and function. Also, the conventional pepsin digestion used for fibrillar collagens cannot be utilized for purifying type IX collagen, because it contains several interruptions in its collagenous triple helix. A baculovirus expression system was used here to produce recombinant human type IX collagen by coinfecting insect cells with three viruses containing full-length cDNAs for the α1(IX), α2(IX), and α3(IX) collagen chains together with a double promoter virus for the α and β subunits of human prolyl 4-hydroxylase. Correctly folded recombinant type IX collagen was secreted, consisting of the three α chains in a 1:1:1 ratio and showing the expected biphasic thermal melting profile. When the individual α chains were expressed, disulfide-bonded homotrimers and homodimers of the α chains were observed. When the cells were coinfected with the viruses for all three α chains, heterotrimers of α1(IX), α2(IX), and α3(IX) were detected in cell culture medium, and the other possible combinations were less prominent. When any two of the α chains were co-expressed, in addition to the homodimers and homotrimers, only α1(IX) and α3(IX) chains were disulfide-bonded. The results thus suggest that the most favored molecular species is an α1(IX)α2(IX)α3(IX) heterotrimer, but the chains are also able to form disulfide-bonded heterotrimers of α1(IX) and α3(IX) chains and (α1(IX))3, (α2(IX))3, and (α3(IX))3 homotrimers.

Recombinant human type II collagen (rhCII) was produced using both the HT1080 mammalian cell expression system (rhCIIht) and a baculovirus expression system (rhCIIbac). The biosynthesis of CII requires extensive post-translational modifications, such as the hydroxylation of prolyl and lysyl residues and glycosylation of hydroxylysyl residues. Amino acid analyses indicated that the rhCIIbac was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived hCII, while rhCIIht was hyperhydroxylated and hyperglycosylated at lysyl residues. When the murine collagen-induced arthritis (CIA) model was used to investigate the immunological properties of the two forms of recombinant CII, each induced a high incidence of arthritis following immunization of susceptible mice when emulsified with complete Freund's adjuvant (CFA). However, the severity of the arthritis, as assessed by the number of affected limbs, was significantly higher in mice immunized with rhCIIht than in mice immunized with rhCIIbac. These data indicate that the degree of hydroxylysine glycosylation may play a role in the induction of the arthritogenic response to CII. Each of the recombinant collagens was comparable to tissue-derived hCII in their ability to induce tolerance and suppress arthritis when given as intravenous or oral tolerogens. Taken together, our data suggest that recombinant CII can be prepared in adequate amounts for therapeutic uses and that the material is immunologically comparable to tissue-derived hCII when used to induce tolerance.

Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [alpha(I)]2beta2, [alpha(II)]2beta2, and [alpha(III)]2beta2 tetramers with identical beta subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic alpha subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.

Collagen is the main structural protein in vertebrates. It plays an essential role in providing a scaffold for cellular support and thereby affecting cell attachment, migration, proliferation, differentiation, and survival. As such, it also plays an important role in numerous approaches to the engineering of human tissues for medical applications related to tissue, bone, and skin repair and reconstruction. Currently, the collagen used in tissue engineering applications is derived from animal tissues, creating concerns related to the quality, purity, and predictability of its performance. It also carries the risk of transmission of infectious agents and precipitating immunological reactions. The recent development of recombinant sources of human collagen provides a reliable, predictable and chemically defined source of purified human collagens that is free of animal components. The triple-helical collagens made by recombinant technology have the same amino acid sequence as human tissue-derived collagen. Furthermore, by achieving the equivalent extent of proline hydroxylation via coexpression of genes encoding prolyl hydroxylase with the collagen genes, one can produce collagens with a similar degree of stability as naturally occurring material. The recombinant production process of collagen involves the generation of single triple-helical molecules that are then used to construct more complex three-dimensional structures. If one loosely defines tissue engineering as the use of a biocompatible scaffold combined with a biologically active agent (be it a gene or gene construct, growth factor or other biologically active agent) to induce tissue regeneration, then the production of recombinant human collagen enables the engineering of human tissue based on a human matrix or scaffold. Recombinant human collagens are an efficient scaffold for bone repair when combined with a recombinant bone morphogenetic protein in a porous, sponge-like format, and when presented as a membrane, sponge or gel can serve as a basis for the engineering of skin, cartilage and periodontal ligament, depending on the specific requirements of the chosen application.

An efficient expression system for recombinant human collagens will have numerous scientific and medical applications. However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity. We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methyl-otrophic yeast Pichia pastoris. These methods are based on co-expression of procollagen polypeptide chains with the α- and β-subunits of prolyl 4-hydroxylase. The triple-helical type-I, -II and -III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins. All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils. The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g/l depending on the collagen type.

Type II collagen is the main structural component of hyaline cartilages where it forms networks of thin fibrils that differ in morphology from the much thicker fibrils of type I collagen. We studied here in vitro the formation of fibrils of pepsin-treated recombinant human type II collagen produced in insect cells. Two kinds of type II collagen preparation were used: low hydroxylysine collagen having 2.0 hydroxylysine residues/1,000 amino acids, including 1.3 glycosylated hydroxylysines; and high hydroxylysine collagen having 19 hydroxylysines/1,000 amino acids, including 8.9 glycosylated hydroxylysines. A marked difference in fibril formation was found between these two kinds of collagen preparation, in that the maximal turbidity of the former was reached within 5 min under the standard assay conditions, whereas the absorbance of the latter increased until about 600 min. The critical concentration with the latter was about 10-fold, and the absorbance/microgram collagen incorporated into the fibrils was about one-sixth. The morphology of the fibrils was also different, in that the high hydroxylysine collagen formed thin fibrils with essentially no interfibril interaction or aggregation, whereas the low hydroxylysine collagen formed thick fibrils on a background of thin ones. The data thus indicate that regulation of the extents of lysine hydroxylation and hydroxylysine glycosylation may play a major role in the regulation of collagen fibril formation and the morphology of the fibrils.

Insect cells coinfected with two baculoviruses, one coding for the pro alpha chains of human type II procollagen and the other for both the alpha and beta subunits of human prolyl 4-hydroxylase, produced the cartilage-specific type II collagen with a stable triple helix. The highest expression levels, up to 50 mg/l of type II collagen, were obtained in suspension culture using a modified construct in which sequences coding for the signal peptide and N propeptide of type II procollagen had been replaced by those for type III procollagen. The type III N propeptide artificially generated into type II procollagen was found to be cleaved at a much higher rate than the wild-type type II N propeptide, probably because the former interacted poorly with the triple-helical domain of type II procollagen. The amino acid composition of the recombinant type II collagen was very similar to that of the non-recombinant protein, but the hydroxylysine content was only 17% and that of glycosylated hydroxylysines was equally low. The hydroxylysine content was increased to the level found in the non-recombinant collagen by using an additional baculovirus coding for lysyl hydroxylase, and a substantial increase was also found in the glycosylated hydroxylysine content. No difference in thermal stability was found between the low- and high-hydroxylysine collagens.