The 100‐kDa chain of nicein/kalinin is a laminin B2 chain variant

Abstract

We have isolated the basement membrane component nicein and performed rotary-shadow analyses using electron microscopy that showed the presence of two forms (I and 11) of the protein. Molecular cloning of the cDNA that codes for the 100-kDa chain of the protein revealed that the sequence matches those independently identified for the 105 -155-kDa subunit of kalinin, a recently identified basement membrane component. These data demonstrate that nicein and kalinin contain an identical chain. The length of the open reading frame in the cDNA (~5 2 0 0 nucleotides) and amino acid sequences obtained from the N-terminus of the 105-kDa kalinin chain showed the occurrence of a precursor polypeptide. This immature polypeptide is probably related to form I, observed by rotary shadowing, while the mature form is related to form II. It is noteworthy that niceinkalinin subunits share discrete sequence similarities with the B2 chain of human laminin, but with a cleavage occurring within domain 111 that eliminates domains IV and V from the final product. The sequence of this subunit is nearly identical to that of B2t, a recently described polypeptide supposed to be related to a new laminin variant. Since niceinkalinin expression is specifically impaired in the severe genodermatosis Herlitz junctional epidermolysis bullosa, the role and structure of this tissue-restricted laminin variant is crucial for the understanding of epidermal-dermal adhesion.The basement membrane (BM) is a type of extracellular matrix which provides an attachment substrate for overlying cell populations and plays a crucial role in organizing tissue architecture, tissue compartmentalization, differentiation of adjacent cells and filtration of nutrients. Since mechanisms mediating these functions are poorly understood, a considerable amount of research has been devoted to the molecular characterization of BM components and elucidation of their biochemical properties. Some differences exist from one type of BM to another and no less than 14 shared macromolecules have already been found [l].We previously reported the isolation of a novel epidermal BM component, called nicein, which is found in various BM species that we investigated with the monoclonal antibody GB3 [2, 31. This glycosylated protein is composed of three major subunits of 100,125 and 150 kDa, as shown by immunoprecipitation assays performed on keratinocytes in culture [3]. Since nicein is secreted by these cells, we isolated the protein by immunoaffinity chromatography from conditioned

CD98hc (SLC3A2) regulation of skin homeostasis wanes with age

“…Formalin-fixed paraffin sections were used for all histology except for CD98, Ki67, 6, and 1 integrins and laminin 332 immunostaining, which were performed on frozen sections. Antibodies were: rabbit antimouse involucrin (Covance), rabbit anti-mouse cytokeratin 10 (Covance), goat anti-mouse Ki67 (M-19; Santa Cruz Biotechnology, Inc.), rat antiintegrin 1 (MAB1997; Millipore), monoclonal anti-6 (GoH3; R&D Systems), rabbit anti-laminin 332 (SE144, gift of L. Gagnoux-Palacios, Institut National de la Sante et de la Recherche Medicale U634, Nice, France; Vailly et al, 1994), rat anti-mouse CD98 (clone RL388; eBioscience), rat antimouse macrophage Mac 2 (clone CL8942 AP; Cedarlane), rat IgG2a isotype control (eBioscience), and Alexa Fluor 488-or 546-conjugated anti-rat, -mouse, -goat, or -rabbit (Invitrogen).…”

Human Hepatocellular Carcinoma (HCC) Cells Require Both α3β1 Integrin and Matrix Metalloproteinases Activity for Migration and Invasion

“…Ln-1 and Coll IV were purchased from Sigma Chemical Company (St. Louis, Missouri). The monoclonal antibody to human Ln-5 (GB3) (Vailly et al, 1994) was a gift of G. Meneguzzi (School of Medicine, Nice, France). The fluorescein isothiocyanate-labeled goat antimouse secondary antibody was purchased from Zymed (San Francisco, California).…”

Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia

“…Synthesis of laminin-5 was monitored using mAb GB3 18 that recognizes native laminin-5, mAb K140 16 and polyclonal antibody (pAb) ␤3B, specific to the laminin ␤3 chain; mAbs BM165 31 and pAb SE85, 32 specific to the laminin ␣3 chain, and pAb SE144, specific to the laminin ␥2 chain 33 were also used. Integrins ␣6 and ␤4, and BP180 were detected using mAbs GoH3, 34 3E1 (Life Technologies) and 233, 35 respectively.…”