Mammalian collagen receptors.

Collagen-rich extracellular matrices are abundant and ubiquitous in the mammalian body. Collagens are not only essential for the mechanical stability of tissues, but are also intimately involved in controlling cell behaviour. The hallmark of collagens is a triple helix made up of polypeptide chains containing glycine-X-Y repeats. A structurally and functionally diverse group of cell surface receptors mediates the recognition of triple-helical collagen: integrins, discoidin domain receptors, glycoprotein VI, leukocyte-associated IG-like receptor-1, and members of the mannose receptor family. In this review, we discuss the structure and function of these receptors, focussing on the principles involved in collagen recognition.     

Quantification of human neutrophil motility in three-dimensional collagen gels. Effect of collagen concentration.

Leukocytes must migrate through tissues to fulfill their role in the immune response, but direct methods for observing and quantifying cell motility have mostly been limited to migration on two-dimensional surfaces. We have now developed methods for examining neutrophil movement in a three-dimensional gel containing 0.1 to 0.7 mg/ml rat tail tendon collagen. Neutrophil-populated collagen gels were formed within flat glass capillary tubes, permitting direct observation with light microscopy. By following the tracks of individual cells over a 13.5-min observation period and comparing them to a stochastic model of cell movement, we quantified cell speed within a given gel by estimating a random motility coefficient (mu) and persistence time (P). The random motility coefficient changed significantly with collagen concentration in the gel, varying from 1.6 to 13.3 x 10(-9) cm2/s, with the maximum occurring at a collagen gel concentration of 0.3 mg/ml. The methods described may be useful for studying tissue dynamics and for evaluating the mechanism of cell movement in three-dimensional gels of extracellular matrix (ECM) molecules.     

Digestion of native collagen, denatured collagen, and collagen fragments by extracts of rat liver lysosomes.

Extracts of highly purified lysosomes from rat liver were examined for their ability to degrade native collagen and thermally denatured collagen at pH values between 3.5 and 7.0. After a 24-h digestion at 36 degrees with the lysosomal extract at a pH of 5.5 or lower (collagen/lysosomal protein; 2/1 or 8/1), both native and denatured collagen were degraded to an extent equivalent to 60 to 70% of that observed upon total acid hydrolysis in 6 N HCl as measured by the ninhydrin reaction (570 nm). At a pH of 6.0, native collagen and denatured collagen were degraded by the mixture of lysosomal proteinases to 11% and 40% of total acid hydrolysis, respectively. At pH 6.5 AND 7.0, the corresponding values were 3% versus 33% and 0.3% versus 11%, respectively. Fragments of collagen (TCA and TCB) are produced when mammalian collagenase degrades native collagen at 25 degrees. These fragments were degraded by the lysosomal extract at 36 degrees to an extent equivalent to 28% and 8% of total acid hydrolysis at pH 6.5 and 7.0, respectively. The experiments at pH 6.5 and 7.0 were done using a collagen/lysosomal protein ratio of 2/1. At pH 5.0 (a pH which is found within secondary lysosomes), the lysosomal extracts degraded collagen to a mixture of free amino acids and small peptides. Amino acid analysis established that approximately 30% of the amino acid residues of the collagen appeared in the lysosomal hydrolysate as free amino acids. Hydroxyproline and perhaps hydroxylysine were the only amino acids found in collagen which did not appear at least to some extent as the free amino acid in this hydrolysate.     

Solubilization of bovine heart-value collagen.

Complete solubilization of bovine heart-valve gollagen was obtained by using sequential pepsin digestion and extraction with dithiothreitol under non-denaturing conditions. Use of the reducing agent was the crucial step resulting in solubilization. These findings suggest that disulphide-bonded proteins may be in part responsible for the insolubility of bovine heart-valve collagen.     

Synthesis of reduced collagen crosslinks.

A new synthetic route to reduced collagen crosslinks (LNL and HLNL) is described in this report. It enables an enantioselective synthesis of LNL. HLNL was obtained as a mixture of two diastereoisomers. This method also provides the possibility to introduce radio-labels during the synthesis.     

Temperature dependence of collagen fluorescence.

Dermal collagens have several fluorescent moieties in the UV and visible spectral regions that may serve as molecular probes of collagen. We studied the temperature-dependence of a commercial calf skin collagen and acid-extracted Skh-1 hairless mouse collagen at temperatures from 9 degrees C to 60 degrees C for excitation/emission wavelengths 270/305 nm (tyrosine), 270/360 nm (excimer-like aggregated species), 325/400 nm (dityrosine) and 370/450 nm (glycation adduct). L-tyrosine (1 x 10(-5) M in 0.5 M HOAc) acted as a "reference compound" devoid of any collagen structural effects. In general, the fluorescence efficiency of these fluorophores decreases with increasing temperature. Assuming that rate constant for fluorescence deactivation has the form k(d)(T) = k(d) degrees exp (-DeltaE/RT), an Arrhenius plot of log[(1/Phi) - 1] vs. 1/T affords a straight line whose (negative) slope is proportional to the activation energy, DeltaE, of the radiationless process(es) that compete with fluorescence. Because it is difficult to accurately measure Phi(f) for collagen-bound fluorophores, we derived an approximate formula for an activation parameter, DeltaE*, evaluated from an Arrhenius-like plot of log 1/I(N)vs. 1/T, (1/I(N)vs. is the reciprocal normalized fluorescence intensity). Tyrosine in dilute solution affords a linear Arrhenius plot in both of the above cases. Using the known value of Phi(f) = 0.21 for free tyrosine at room temperature, we determined that DeltaE* is accurate to approximately 25% in the present instance. Collagen curves are non-linear, but they are quasi-linear below approximately 20 degrees C, where the helical form predominates. Values of DeltaE* determined from the data at T < 20 degrees C ranged from 6.2-8.4 kJ mol(-1) (1.5-2.0 kcal mol(-1)) for mouse collagen and 10.3-11.4 kJ mol(-1) (2.5-2.7 kcal mol(-1)) for calf skin collagen, consistent with collisional deactivation of the fluorescent state via thermally enhanced molecular vibrations and rotations. Above 20 degrees C, log 1/I(N)vs. 1/T plots from Skh-1 hairless mouse collagen are concave-downward, suggesting that fluorescence deactivation from the denatured coil has a significant temperature-independent component. For calf skin collagen, these plots are concave-upward, suggesting an increase in activation energy above Tm. These results suggest that collagen backbone and supramolecular structure can influence the temperature dependence of the bound fluorophores, indicating the future possibility of using activation data as a probe of supramolecular structure and conformation.     

Reconstituted collagen fibrils. Fibrillar and molecular stability of the collagen upon maturation in vitro.

During the maturation in vitro of reconstituted collagen fibrils prepared from rat skin, the mechanical and thermal stability of collagen increased and the pepsin-solubility decreased. At the same time a larger fraction of the pepsin-soluble collagen attained a lower molecular thermal stability that resulted in a biphasic thermal transition of the soluble collagen. Type-I collagen, with a similar biphasic thermal transition, was isolated from acid-insoluble rat skin collagen.     

The collagen of heart valve.

A hydroxylysine-rich type I collagen has been isolated from pepsin-digested porcine heart valve. The ratio of alpha1 to alpha2 in the isolated molecule was 2:1. The component alpha chains exhibited unusual chromatographic behavior in comparison to corresponding chains from human dermis and lathyritic rat skin collagen. The composition of component cyanogen bromide peptides identified the alpha chains as authentic type I chains and demonstrated hydroxylysine enrichment throughout the length of the chain. delta6-Dehydro-5,5'dihydroxylysinonorleucine, a collagen cross-link derived from two hydroxylysyl residues and ordinarily found in hard tissue collagens was found to be the predominant cross-link in heart valve.     

Radiation-induced aldehydes in collagen.

Aldehydes present in native and irradiated (30 krad) collagen were separated from its enzymic degradation products using molecular-sieve chromatography on Bio-Gel P-2 column. From the absorption spectra of N-methylbenzothiazolone hydrazone derivatives it was concluded that two out of five of the aldehydes separated from the irradiated collagen, were identical with those present in control collagen. The possible origin of three other aldehydes is discussed.     

Relaxin and collagen metabolism.

The influence of the peptide hormone relaxin on collagen metabolism was studied in the symphysis pubis of the mouse. In the tissue the content of water and of acid soluble collagen in relation to total collagen is increased by hormonal treatment. Total collagen calculated in relation to the dry weight is decreased. Collagenase which was also detected in the symphyses of the controls is slightly enhanced. In serum collagen peptidase and collagen peptidase inhibitor as well as cyclic AMP exhibit distinctly increased levels. The effects can be suppressed by administration of relaxin-specific antisera. The data make clear that in the symphysis relaxin activates the collagenolytic system.     

Occurrence of collagen and proteoglycan forms of type IX collagen in chick embryo cartilage. Production and characterization of a collagen form-specific antibody.

Type IX collagen from chick embryonic cartilage is a proteoglycan bearing a single chondroitin sulfate chain covalently linked to the alpha 2(IX) polypeptide chain. We have isolated type IX collagen metabolically labeled with [3H]proline using an antibody to type IX collagen and have found that the molecule is synthesized in two forms, a collagen form (COLIX) and a proteoglycan form (PGIX). In cultured chondrocytes, the two forms of type IX collagen showed a different ability to be deposited in the matrix. We have suggested the possibility that both forms may arise from an alternative substitution of a chondroitin sulfate chain to the NC3 domain of the alpha 2(IX) chain. Based on the reported amino acid sequence at the NC3 domain of alpha 2(IX), we have synthesized undecapeptides containing the sequence around the glycosaminoglycan attachment site of the alpha 2(IX) chain. Antibody against the peptide, which was raised in rabbit, only recognized COLIX and made it possible to distinguish COLIX from PGIX. Evidence shows that this could be due to a difference in antigenicity of the NC3 domain of the alpha 2(IX) chain between COLIX and PGIX caused by the substitution of a chondroitin sulfate chain to the serine residue in this domain. Therefore, this antibody may be useful as a probe for studies on the functions of glycosaminoglycan substitution in type IX collagen.     

Biosynthesis of collagen crosslinks. II. In vivo labelling and stability of lung collagen in rats

Rat lung collagen was labelled in vivo by a single intraperitoneal injection of [3H]lysine at several key timepoints in lung development: days 11 (alveolar proliferation), 26 (start of equilibrated growth), 42 (end of equilibrated growth), and 100 (adult lung structure present). The rates of deposition of labelled hydroxylysine and the difunctional, Schiff base-derived crosslinks hydroxylysinonorleucine (HLNL) and dihydroxylysinonorleucine (DHLNL) were quantified. We also measured total lung content of the trifunctional, mature crosslink hydroxypyridinium (OHP) in these same animals. While the relative rates of accumulation of labelled collagen [3H]hydroxylysine differed by a factor of about 6 at the different times of injection of labelled precursor, quantitative and qualitative patterns of collagen crosslinking were very similar at all of the lung developmental stages studied. Furthermore, there was little or no breakdown of the lung collagen pool as defined by the presence of labelled crosslinks; changes in lung DHLNL content could be completely accounted for by its maturation to OHP, regardless of the age of the rats when injected with the radioactive precursor. We conclude that mature, crosslinked collagen in the lungs of rats, which is obligatorily an extracellular pool, is not being degraded at a measurable rate. Therefore, studies of others that have shown apparent high rates of breakdown of newly synthesized collagen in lungs of whole animals using different methods are probably not reflective of the metabolic fate of total lung collagen, and may indicate that degradation of normal lung collagen occurs predominantly or exclusively intracellularly.     

Procoagulant activity of collagen. Effect of difference in type and structure of collagen

Procoagulant activities of different types and structures of collagen were examined. Collagens used were types I (including its methylated and succinylated forms), II, III, IV and V. Each collagen was coated on an inner surface of a glass tube. The change of fluidity during coagulation of blood in the tube was measured by means of a new rheological technique. For monomeric collagen, the procoagulant activity of the succinylated form (negatively charged) was higher than that of the methylated form (positively charged). The procoagulant activity of type IV (dry) was lower than that of other types of collagen. For fibrillar collagens, the initiation of coagulation for type V (non-banded) was fairly delayed compared to those for types I, II and III (banded). An increase in water content in both monomeric and fibrillar forms promoted procoagulant activity. For most of the collagen forms, the addition of factor XII inhibitor (Polybrene) to blood brought about a remarkable delay of the initiation of coagulation, suggesting that the activation of factor XII on the collagen surface is one of main factors governing procoagulant activity. In addition, our data suggest that large numbers of adherent platelets to the collagen surface promote activation of the intrinsic coagulation system.     

Discrete integration of collagen XVI into tissue-specific collagen fibrils or beaded microfibrils.

The structural and functional diversity of extracellular matrices is determined, not only by individual macromolecules, but even more decisively, by the alloyed aggregates they form. Although quantitatively major matrix molecules can occur ubiquitously, their organization varies from one tissue to another due to their amalgamation with specific sets of minor components. Here, we show that the fibril-associated collagen with interrupted triple helices collagen XVI is unique in that, depending on the tissue context, it can be incorporated into distinct suprastructural aggregates. In papillary dermis, the protein unexpectedly does not occur in banded collagen fibrils, but rather, is a component of specialized fibrillin-1-containing microfibrils. In territorial cartilage matrix, however, collagen XVI is not a component of aggregates containing fibrillin-1. Instead, the protein resides in a discrete population of thin, weakly banded collagen fibrils also containing collagens II and XI. Collagen IX also occurs in this population of fibrils, but at longitudinal locations discrete from those of collagen XVI. This suprastructural versatility of a collagen is without precedent and highlights pivotal differences in the tissue-specific organization of matrix aggregate structures.     

Subcellular localization of transglutaminase. Effect of collagen.

1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5'-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.