Molecular cloning and expression of a novel keratinocyte protein (psoriasis-associated fatty acid-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin and that shares similarity to fatty acid-binding proteins.
Analysis by means of two-dimensional (2D) gel electrophoresis of the protein patterns of normal and psoriatic unfractionated non-cultured keratinocytes has revealed a few low-molecular-weight proteins that are highly up-regulated in psoriatic skin. These include psoriasin; calgranulin B, also known as MRP 14, L1, or calprotectin; calgranulin A or MRP 8; and cystatin A or stefin A. Here, we have cloned and sequenced the cDNA (clone 1592) encoding a new member of this group of low-molecular-weight proteins [isoelectric focusing (IEF) SSP 3007 in the keratinocyte 2D gel protein database] that we have termed PA-FABP (psoriasis-associated fatty acid-binding protein). The deduced sequence predicted a protein with molecular weight of 15,164 daltons and a calculated pI of 6.96, values that are close to those recorded in the keratinocyte 2D gel protein database. The protein comigrated with PA-FABP as determined by 2D gel analysis of [35S]-methionine-labeled proteins expressed by transformed human amnion (AMA) cells transfected with clone 1592 using the vaccinia virus expression system and reacted with a rabbit polyclonal antibody raised against 2D gel purified PA-FABP. Structural analysis of the amino acid sequence revealed 48%, 52%, and 56% identity to known low-molecular-weight fatty acid-binding proteins belonging to the FABP family. Northern blot analysis showed that PA-FABP mRNA is indeed highly up-regulated in psoriatic keratinocytes. The transcript is present in human cell lines of epithelial and lymphoid (Molt 4) origin but cannot be detected in normal or SV40 transformed MRC-5 fibroblasts. 2D gel protein analysis of normal primary keratinocytes cultured for at least 8 d under conditions that promoted incomplete terminal differentiation [serum-free keratinocyte (SFK) medium supplemented with epidermal growth factor (EGF), pituitary extract, and 10% fetal calf serum] revealed a strong up-regulation of PA-FABP, psoriasin, calgranulins A and B, and a few other proteins that are highly expressed in psoriatic skin. The levels of these proteins exceeded by far those observed in non-cultured normal keratinocytes implying that the cultured cells have followed an altered pattern of differentiation that resembles--at least in part--that of non-cultured psoriatic keratinocytes. The implications of these results for the study of psoriasis are discussed.
Madsen
P
P
Institute of Medical Biochemistry, Aarhus University, Denmark.
Rasmussen
H H
HH
Leffers
H
H
Honoré
B
B
Celis
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000032:analysis; D040501:Calgranulin A; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D011565:Psoriasis / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation
D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D002352:Carrier Proteins; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D025521:Tumor Suppressor Proteins