Abstract. Previous work from our laboratories has demonstrated that: (a) the striated collagen fibrils of the corneal stroma are heterotypic structures composed of type V collagen molecules coassembled along with those of type I collagen, (b) the high content of type V collagen within the corneal collagen fibrils is one factor responsible for the small, uniform fibrillar diameter (25 nm) characteristic of this tissue, (c) the completely processed form of type V collagen found within tissues retains a large noneollagenous region, termed the NH2-terminal domain, at the amino end of its txl chain, and (d) the NH2-terminal domain may contain at least some of the information for the observed regulation of fibril diameters. In the present investigation we have employed polyclonal antibodies against the retained NH2-terminal domain of the ot 1 (V) chain for immunohistochemical studies of embryonic avian corneas and for immunoscreening a chicken cDNA library. When combined with cDNA sequencing and molecular rotary shadowing, these approaches provide information on the molecular structure of the retained NH2-terminal domain as well as how this domain might function in the regulation of fibrillar structure.In immunofluorescence and immunoelectron microscopy analyses, the antibodies against the NH2-terminal domain react with type V molecules present within mature heterotypic fibrils of the corneal stroma. Thus, epitopes within at least a portion of this domain are exposed on the fibril surface. This is in marked contrast to mAbs which we have previously characterized as being directed against epitopes located in the major triple helical domain of the type V molecule. The helical epitopes recognized by these antibodies are antigenically masked on type V molecules that have been assembled into fibrils. Sequencing of the isolated eDNA clones has provided the conceptual amino acid sequence of the entire amino end of the cd(V) procollagen chain. The sequence shows the location of what appear to be potential propeptidase cleavage sites. One of these, if preferentially used during processing of the type V procollagen molecule, can provide an explanation for the retention of the NH2-terminal domain in the completely processed molecule. The sequencing data also suggest that the NH2-terminal domain consists of several regions, providing a structure which fits well with that of the completely processed type V molecule as visualized by rotary shadowing.T HE major component of the mature corneal stroma is a striated collagen fibril characterized by its small, uniform diameter (25 /tm). Because these fibrillar properties are thought to be required for corneal transparency, elucidating the molecular mechanism(s) by which they are controlled is paramount to our understanding of the development and growth of a functional cornea, as well as of fibrillogenesis in general.

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific "unmasking" treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined, only Bowman's membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the "unmasking" did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.Within both developing embryos and adult organisms, most cells and tissues are intimately associated with some form of collagenous extracellular matrix. Such matrices fall into two general categories: (a) the stromas, which generally become populated with mesenchymal cells and consist largely of extracellular materials having structural and supportive roles within tissues and organs, and (b) the basement membranes, which are aceUular sheets found at all epithelial-mesenchymal interfaces and surrounding some types of individual cells. The collagenous components of these matrices constitute a hetero-

Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of ϳ50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F112F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.Basement membranes, a class of extracellular matrices which separate epithelia from connective tissues and surround some types of cells such as smooth and skeletal muscle, have been implicated in mediating cell and tissue interactions during embryonic development, tissue repair, and regeneration (for reviews, see references 1 and 2). Evaluation of the proposed roles for these structures requires specific and sensitive probes for identification, localization, and manipulation of their various molecular components. One such probe is the monoclonal antibody (3-5), which can be selected not only for specificity to an individual molecular component, but for specificity to an individual domain within a molecule.Type IV collagen is a major component of many basement membranes. Recent work on the mouse EHS-sarcoma (6), chicken gizzard (7), human placenta (8), and bovine lens capsule (9) have provided some general information about the structure of the molecule (reviewed in references 10 and 11). The native type IV collagen molecule is composed of three chains (each ~ 180,000 mol wt) representing at least two different gene products. These are assembled so that each molecule contains one or more triple-helical regions (collagenous domains) and at least two regions in which the chains are not folded into the triple-helical conformation (noncollagenous domains). A model has been proposed to explain how type IV molecules may be arranged as supramolecular aggregates within basement membranes (12, 13). Its salient features have been inferred from specific enzymatic digestions coupled with electron microscopic observations of rotary shadowed preparations. The model propo...

In the cartilaginous epiphyseal growth plate, extracellular matrix molecules such as collagens are believed to play important roles during both normal and abnormal development. One defect of the epiphyseal plate occurs in chickens with a condition termed tibial dyschondroplasia (TD). This abnormality occurs in certain strains of juvenile chickens and other rapidly developing animals. It is characterized by the presence of a mass of avascular, uncalcified cartilage which is retained in the proximal metaphysis of the tibiotarsus. To elucidate the developmental events which may be involved in this lesion, we have performed both immunohistochemistry and in situ hybridizations for collagen types I1 and X, known components of the extracellular matrix of the growth plate. By immunohistochemical analyses, the TD lesion contains both of these collagen types; therefore, the presence of these molecules per se is not sufficient for calcification of vascularization to occur. Since type X collagen is expressed exclusively in hypertrophic cartilage, the chondrocytes in the lesion must have undergone hypertrophy before their developmental arrest. The matrix of the lesion also reacted with a monoclonal antibody which is directed against an epitope in the NH,-terminal telopeptide of the al(I1) chain. Our previous data suggest that this epitope is rendered unavailable in type 11 collagen which has undergone crosslink formation; its availability in the lesion suggests that crosslinking may be abnormal. Lastly, analyses by in situ hybridization failed to detect mRNA for either type I1 or type X collagen within the lesion, but chondrocytes distal to the lesion do contain mRNAs for these collagens in a spatial pattern suggesting the presence of a second growth plate. 0 1993 Wiley-Liss, Inc.