Holger Notbohm

Characterization of Human Type III Collagen Expressed in a Baculovirus System PRODUCTION OF A PROTEIN WITH A STABLE TRIPLE HELIX REQUIRES COEXPRESSION WITH THE TWO TYPES OF RECOMBINANT PROLYL 4-HYDROXYLASE SUBUNIT

An efficient expression system for recombinant collagens would have numerous scientific and practical applications. Nevertheless, most recombinant systems are not suitable for this purpose, as they do not have sufficient amounts of prolyl 4-hydroxylase activity. Pro-α1 chains of human type III collagen expressed in insect cells by a baculovirus vector are reported here to contain significant amounts of 4-hydroxyproline and to form triple-helical molecules, although the T of the triple helices was only about 32-34°C. Coexpression of the pro-α1(III) chains with the α and β subunits of human prolyl 4-hydroxylase increased the T to about 40°C, provided that ascorbate was added to the culture medium. The level of expression of type III procollagen was also increased in the presence of the recombinant prolyl 4-hydroxylase, and the pro-α1(III) chains and α1(III) chains were found to be present in disulfide-bonded molecules. Most of the triple-helical collagen produced was retained within the insect cells and could be extracted from the cell pellet. The highest expression levels were obtained in High Five cells, which produced up to about 80 μg of cellular type III collagen (120 μg of procollagen) per 5 × 106 cells in monolayer culture and up to 40 mg/liter of cellular type III collagen (60 mg/liter procollagen) in suspension. The 4-hydroxyproline content and T of the purified recombinant type III collagen were very similar to those of the nonrecombinant protein, but the hydroxylysine content was slightly lower, being about 3 residues/1000 in the former and 5/1000 in the latter.

High‐level production of human type I collagen in the yeast Pichia pastoris

Four human genes, two of them encoding the proα1 and proα2 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 proα1 and proα2 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 proα 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. Copyright

Recombinant Human Type II Collagens with Low and High Levels of Hydroxylysine and Its Glycosylated Forms Show Marked Differences in Fibrillogenesis in Vitro

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.

Expression of Wild-Type and Modified Proα Chains of Human Type I Procollagen in Insect Cells Leads to the Formation of Stable [α1(I)]2α2(I) Collagen Heterotrimers and [α1(I)]3 Homotrimers but Not [α2(I)]3 Homotrimers

Insect cells coinfected with a baculovirus coding for the proα1(I) chain of human type I procollagen and a double promoter virus coding for the α and β subunits of human prolyl 4-hydroxylase produced homotrimeric [proα1(I)]3procollagen molecules. The use of an additional virus coding for the proα2(I) chain led to the formation of a heterotrimeric molecule with the correct 2:1 ratio of proα1 to proα2 chains of type I procollagen (proα1(I) and proα2(I) chains, respectively), unless the proα1(I) chain was expressed in a relatively large excess. Replacement of the sequences coding for the signal peptide and the N propeptide of the proα1(I) chain with those of the proα1(III) chain increased level of expression of the proα1(I) chain, whereas no similar effect was found when the corresponding modification was made to the virus coding for the proα2(I) chain. Molecules containing such modified N propeptides were found to be processed at their N terminus more rapidly than those containing the wild-type propeptides. TheT m of the type I collagen homotrimer was similar to that of the heterotrimer, both values being about 42–43 °C when determined by circular dichroism. The wild-type proα2(I) chain formed no homotrimers. Replacement of the C propeptide of the proα2(I) chain with that of the proα1(I) chain or proα1 chain of type III procollagen (proα1(III) chain) led to the formation of homotrimers, but the α2(I) chains in such molecules were completely digested by pepsin in 1 h at 22 °C. The data thus suggest that, in addition to control at the level of the C propeptide, other restrictions may exist at the level of the collagen domain that prevent the formation of stable homotrimeric [proα2(I)]3 molecules in insect cells.

Chondrocyte redifferentiation in 3D: The effect of adhesion site density and substrate elasticity †

To obtain sufficient cell numbers for cartilage tissue engineering with autologous chondrocytes, cells are typically expanded in monolayer culture. As a result, they lose their chondrogenic phenotype in a process called dedifferentiation, which can be reversed upon transfer into a 3D environment. We hypothesize that the properties of this 3D environment, namely adhesion site density and substrate elasticity, would influence this redifferentiation process. To test this hypothesis, chondrocytes were expanded in monolayer and their phenotypical transition was monitored. Agarose hydrogels manipulated to give different RGD adhesion site densities and mechanical properties were produced, cells were incorporated into the gels to induce redifferentiation, and constructs were analyzed to determine cell number and extracellular matrix production after 2 weeks of 3D culture. The availability of adhesion sites within the gels inhibited cellular redifferentiation. Glycosaminoglycan production per cell was diminished by RGD in a dose-dependent manner and cells incorporated into gels with the highest RGD density, remained positive for collagen type I and produced the least collagen type II. Substrate stiffness, in contrast, did not influence cellular redifferentiation, but softer gels contained higher cell numbers and ECM amounts after 2 weeks of culture. Our results indicate that adhesion site density but not stiffness influences the redifferentiation process of chondrocytes in 3D. This knowledge might be used to optimize the redifferentiation process of chondrocytes and thus the formation of cartilage-like tissue.

Expression and characterization of recombinant human type II collagens with low and high contents of hydroxylysine and its glycosylated forms.

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.

The acidic C‐terminal domain of protein disulfide isomerase is not critical for the enzyme subunit function or for the chaperone or disulfide isomerase activities of the polypeptide

Protein disulfide isomerase (PDI) is a multifunctional polypeptide that acts as a subunit in the animal prolyl 4‐hydroxylases and the microsomal triglyceride transfer protein, and as a chaperone that binds various peptides and assists their folding. We report here that deletion of PDI sequences corresponding to the entire C‐terminal domain c, previously thought to be critical for chaperone activity, had no inhibitory effect on the assembly of recombinant prolyl 4‐hydroxylase in insect cells or on the in vitro chaperone activity or disulfide isomerase activity of purified PDI. However, partially overlapping critical regions for all these functions were identified at the C‐terminal end of the preceding thioredoxin‐like domain a′. Point mutations introduced into this region identified several residues as critical for prolyl 4‐hydroxylase assembly. Circular dichroism spectra of three mutants suggested that two of these mutations may have caused only local alterations, whereas one of them may have led to more extensive structural changes. The critical region identified here corresponds to the C‐terminal α helix of domain a′, but this is not the only critical region for any of these functions.

Histologic analysis of thermal effects of laser thermokeratoplasty and corneal ablation using Sirius-red polarization microscopy

Purpose: To evaluate how well several histologic techniques differentiate degrees of thermally induced changes in corneal tissue after laser thermokeratoplasty (LTK) or corneal ablation. Setting: Medical Laser Center Lübeck, Germany. Methods: Corneas of freshly enucleated porcine eyes were treated with a continuous wave laser diode (1.86 &mgr;m) and a pulsed chromium‐thulium‐holmium:YAG laser (2.1 &mgr;m) to produce LTK lesions or ablated with a Q‐switched and a free‐running chromium‐erbium:YSGG laser (2.70 &mgr;m), a free‐running erbium:YAG laser (2.94 &mgr;m), and an argon‐fluoride excimer laser (193 nm). The lesions were evaluated by light microscopy (LM) (hematoxylin and eosin, Azan, van Gieson’s, and Masson‐Goldner’s trichrome stains), transmission electron microscopy (TEM), and polarization microscopy after Sirius‐red staining. Sirius‐red, a strongly elongated, birefringent molecule binding parallel to collagen molecules, was used to enhance corneal birefringence. Results: With routine LM, it was difficult to discriminate the degrees of thermal alterations in LTK lesions. Combined Sirius‐red staining and polarization microscopy distinguished between a strongly coagulated central zone and the transition zone to normal tissue. Sirius‐red uptake was increased in both zones, reflecting the availability of new binding sites. The central zone appeared darker under polarization than normal collagen because of a loss of birefringence. Intrinsic birefringence was greatly reduced; however, form birefringence partly remained as long as some collagen fibrils were intact. In the center of very strong lesions, where the collagen was hyalinized, birefringence was completely lost because of the complete disintegration of the fibrillar structure, which was visible under TEM. The transition zone toward normal cornea showed increased birefringence because the natural birefringence was largely preserved and enhanced by the increased Sirius‐red uptake. Mechanical stretching between neighboring LTK lesions was manifested by increased birefringence. Conclusion: Sirius red offered an improved and simple histologic method for analyzing thermal collagen changes. It may contribute to a better understanding of the working mechanisms of LTK and improve analysis of thermal effects in corneal ablation.

Changes with age in the urinary excretion of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP).

There is increasing evidence that the measurement of urinary hydroxylysylpyridinoline (HP or PYD) and lysylpyridinoline (LP or DPD) by HPLC (high performance liquid chromatography) is potentially useful in clinical and pharmacological studies. HP and LP are promising markers of bone resorption because their levels in urine reflect the breakdown of mature collagen fibrils mainly of skeletal tissues. HP and LP are two non-reducible cross-links of mature collagen which are formed by a sequence of post-translational modifications. HP is a derivative of three residues of hydroxylysine and is present in almost all mature tissues (e.g. tendon. vessel walls, cartilage, dentine and bone). LP is a derivative of two residues of hydroxylysine and one residue of lysine and is present mainly in dentine and bone. Neither cross-link is found in normal human skin. We have isolated and purified HP and LP from commercially available bone gelatine by a preparative reverse-phase column HPLC. These two components were used as external standards for sample analysis. In the present study we analysed the urinary excretion of HP and LP in a group of 264 male and 279 female healthy subjects aged from 6 months to 65 years. A continuous decline of both cross-link components during childhood paralleled by a decrease of the HP:LP-ratio was observed. The levels of HP and LP were 2.5-5 times higher in infants (0.5-1 year) than in children (5-10 years) and 15-20 times higher than in adults (26-65 years). After the age of 17 years, both parameters remained at low levels. These data allow a precise quantitative monitoring of bone resorption in patients with metabolic bone diseases or during pharmacological interventions.

In vitro formation and aggregation of heterotypic collagen I and III fibrils

In vitro fibrillogenesis of solutions containing pepsin digested and acid soluble collagens I and III from human and bovine skin were investigated by turbidity-time measurements, dark-field and electron microscopy. The maximum turbidity of these solutions exhibited inversely proportional dependence on the collagen III content. Self-assembly was accelerated by collagen III. As a measure of mass per unit length, the maximum turbidity shows a mean decrease of 88% when comparing the absorbance at 313 nm for 0% and 50% collagen III in a composite solution of acid extracted collagen. In contrast to these findings, the diameter of fibrils from acid extracted fetal calf skin with 50% collagen III, determined from electron micrographs, was only 23% smaller than for pure collagen I. Correspondence with investigations on in vitro fibrillogenesis with dark-field microscopy and electron microscopy, this phenomenon apparently derives from the bundling of fibrils. This may be interpreted to mean that bundling of fibrils is already suppressed at low collagen III concentrations. A comparison of acid and pepsin extracted fetal calf skin yielded similar behaviour of collagen I and III mixtures, even though the pepsin extract displayed a turbidity reduction that was about 25% less than the acid extract. For pepsin digested collagen from human and bovine skin, differences were found for maximum turbidity and the ability to form bundles decreasing with the biological age of the donor.