heparitin-sulfate and Pemphigoid--Bullous

On histologic vertical sections of skin, the epidermis is separated from the dermis by an amorphous thin membrane, the basal lamina. Ultrastructurally, the basal lamina is composed of four areas, including the basal-cell plasma membrane and hemidesmosomes, the lamina lucida, the lamina densa, and the sub-lamina densa fibrillar region. In culture, epidermal keratinocytes are able to produce hemidesmosomes, lamina lucida, and lamina densa. There is no evidence that cultured keratinocytes can produce sub-lamina densa fibrils. Biochemically, the lamina lucida contains two major glycoproteins. One, the bullous pemphigoid antigen, is synthesized by epidermal keratinocytes in vitro. These cells also synthesize laminin, the other glycoprotein of lamina lucida. At the interface between lamina lucida and lamina densa there is probably a heparan sulfate proteoglycan. Whether this proteoglycan is produced by keratinocytes in culture is not known, but the possibility can be considered. Lamina densa contains collagen IV, and this collagen is synthesized by keratinocytes in culture. However, cultured keratinocytes may also synthesize collagen types I, III, and V. Type V is associated with the basal lamina, but its exact location is unknown. Types I and III (if they are produced in vivo) would be situated in the sub-basal lamina region. The problem of fibronectin remains unsolved. There is "some" fibronectin in the lamina lucida, but its origin is not clear.

Topics: Animals; Basement Membrane; Epidermal Cells; Fibronectins; Glycoproteins; Heparitin Sulfate; Keratins; Laminin; Microscopy, Electron; Pemphigoid, Bullous; Protein Biosynthesis; Swine

Topics: Animals; Antigens; Basement Membrane; Collagen; Fibronectins; Glycoproteins; Heparitin Sulfate; Humans; Laminin; Membrane Proteins; Mice; Molecular Weight; Pemphigoid, Bullous; Skin

Anti-p200 pemphigoid is an autoimmune subepidermal blistering disease characterized by autoantibodies to a 200-kDa protein (p200) of the dermal-epidermal junction (DEJ). p200 has been demonstrated to be distinct from all major DEJ autoantigens and is thought to be important for cell-matrix adhesion. This study provides the first biochemical characterization of p200. Differential extraction experiments demonstrated that efficient recovery of p200 from the dermis was strongly dependent on the presence of reducing agents, suggesting that it forms highly insoluble oligomers and/or is extensively cross-linked to other extracellular matrix components by disulfide bonding. p200 was resistant to digestion with bacterial collagenase, whereas this treatment did degrade major collagenous proteins of the dermis, including type I, VI, and VII collagen. This finding firmly established the noncollagenous nature of p200. N-Glycosidase F reduced the molecular size of the p200 autoantigen from 200 to 190 kDa without decreasing its immunoreactivity. In contrast, digestion of p200 with neuraminidase, O-glycosidase, chondroitinase ABC, and heparitinase I had no effect on its electrophoretic mobility. These data suggest that the p200 molecule contains N-glycans but lacks O-linked oligosaccharides and chondroitin/heparan sulfate side chains. Two-dimensional gel electrophoresis demonstrated that p200 is an acidic protein with an isoelectric point of 5.4 to 5.6. Six different p200-specific sera recognized an identical protein spot of two-dimensionally separated dermal extracts, confirming that patients with this novel autoimmune disease indeed form a single pathobiochemical entity.

Topics: Acids; Autoantigens; Basement Membrane; Buffers; Cells, Cultured; Chondroitin Sulfates; Collagen; Dermis; Disulfides; Electrophoresis, Gel, Two-Dimensional; Epidermis; Fibroblasts; Glycoproteins; Glycosylation; Heparitin Sulfate; Humans; Keratinocytes; Mass Spectrometry; Molecular Weight; Pemphigoid, Bullous; Sequence Analysis, Protein; Solubility; Urea

Specific antisera were used to investigate the distributions of several basement membrane zone (BMZ) components, namely, bullous pemphigoid antigen (BPA), heparan sulfate proteoglycan (HSPG), laminin, and type IV collagen, during the development of hair follicles in late embryo rats. BPA was not visible by indirect immunofluorescence in the BMZ before epidermal involution but appeared in all regions of BMZ after this had occurred. As follicular length increased during maturation, the distribution of BPA was no longer uniform, being reduced or absent from the BMZ around the lower part of the elongating follicle. HSPG was associated with the basal cell layer prior to the appearance of hair follicle primordia and became BMZ-associated before birth but after follicle buds were first observed. HSPG was also found to be associated with the basal cell surfaces in the epidermis, but not in the hair follicle. Laminin and type IV collagen were continually present in epidermal and follicular BMZ both before and during development of hair follicles and were later present in the dermal papilla matrix. From these observations we conclude that (1) laminin and type IV collagen are functionally important for BMZ integrity before and during epidermal and follicular maturation, (2) HSPG may have a cell surface function in epidermis as well as roles in BMZ organization and properties, and (3) the distribution of BPA is indicative of its association only with regions of tissue not involved in morphogenetic change. We also suggest that the cell-matrix interactions documented for BPA, HSPG, laminin, and fibronectin may depend on the type of tissue involved and its state of development, differentiation, or repair.

Topics: Animals; Antigens; Basement Membrane; Chondroitin Sulfate Proteoglycans; Collagen; Female; Fluorescent Antibody Technique; Hair; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immune Sera; Laminin; Male; Morphogenesis; Pemphigoid, Bullous; Pregnancy; Rats; Rats, Inbred Strains; Skin

Adult human skin was separated at the dermal-epidermal junction (DEJ) by 4 published methods that involved different mechanisms of action: cold 1 M salt (tissue extraction), cold trypsinization (enzymatic), induction of a suction blister (mechanical), and warm phosphate-buffered saline (protease activation). The localization of DEJ macromolecules was studied after each separation method. By all of the methods tested, bullous pemphigoid antigen remained closely associated with the epidermis while laminin, the basement membrane heparan sulfate proteoglycan, and collagen types IV and V remained with the dermal side of the separation. The bullous pemphigoid antigen is, then, the DEJ component most closely associated with the epidermal basal cell. Of the basement membrane components tested, only the basement membrane heparan sulfate proteoglycan was trypsin-sensitive.

Topics: Adult; Antigens; Basement Membrane; Collagen; Fluorescent Antibody Technique; Heparitin Sulfate; Humans; Pemphigoid, Bullous; Skin

Progress has been made in identifying and characterizing basement membrane macromolecules, including type IV collagen, laminin, a heparan sulfate proteoglycan and bullous pemphigoid antigen. Basement membrane contains a unique collagen, type IV collagen, which is formed of pro alpha 1(IV) (Mr = 185,000) and pro alpha 2(IV) (Mr = 170,000) chains. As opposed to the fibrillar pattern seen with other collagens, the type IV collagen molecules are thought to be arranged in a honey-comb or reticular pattern which provides the major structural element of the basement membrane. Consistent with this model, type IV collagen has been localized to the basement membrane lamina densa, a nonfibrillar structure. Laminin is a large (Mr = 1,000,000) noncollagenous glycoprotein with chains of 200,000 and 400,000 daltons. It has been localized to the basement membrane lamina lucida and functions to bind epithelial cells to the basement membrane. A heparan sulfate proteoglycan has also been identified in the basement membrane. Its biological function may be to restrict the penetration of anionic macromolecules through the basement membrane. In contrast to the above-mentioned components which are found in all tissue basement membranes, bullous pemphigoid antigen is only found in certain basement membranes, mostly those of stratified squamous epithelia. Bullous pemphigoid antigen is a protein, synthesized by keratinocytes in culture, with disulfide-linked chains (Mr = 220,000). By immunoelectron microscopy, it is localized in the lamina lucida of epidermal basement membrane and is closely associated with the basal cell surface. Its biological function is not known, but could involve epidermal basal cell-substrate interactions which occur when basal cells re-epithelialize wounds.

Topics: Animals; Antigens; Basement Membrane; Collagen; Glycoproteins; Guinea Pigs; Heparitin Sulfate; Humans; Laminin; Molecular Weight; Pemphigoid, Bullous; Skin