Human keratinocytes adhere to multiple distinct peptide sequences of laminin.
In normal human skin, basal layer keratinocytes of the epidermis are intimately associated with the lamina lucida of the basement membrane. Laminin, which is an 850-kD glycoprotein that has a cruciform shape by rotary shadowing and electron microscopy, is localized to the lamina lucida. The present study was aimed at further characterizing the interaction between laminin and cultured human keratinocytes. Initial studies revealed that laminin-coated substrata significantly promoted keratinocyte attachment in a concentration-dependent manner. To further define keratinocyte binding regions within laminin, a 440-kD proteolytic fragment of laminin was generated by limited chymotrypsin digestion, which renders laminin devoid of all terminal globular domains. Substrata coated with this 440-kD laminin fragment did not promote keratinocyte adhesion, suggesting that the globular domains may play an important role in cell adhesion. Based on these experiments, a series of chemically synthesized peptides derived from the A or B1 chains of laminin were studied. Among these, three peptides were found to be active in directly promoting keratinocyte adhesion: peptide F-9 (RYVVLPRPVCFEK) from the inner globule of the human B1 chain, TG-1 (RPVRHAQCRVCDGNSTNPRERH) from the top globule of the amino terminus (short arm) of the A chain, and GD-6 (KQNCLSSRASFRGCVRNLRLSR) from the large carboxy terminal globule at the end of the long arm of the A chain. In competition assays, these peptides in solution were shown to inhibit laminin-mediated keratinocyte adhesion. These studies show that normal human keratinocytes bind directly to laminin at a minimum of three distinct sites.
Wilke
M S
MS
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis 55455-0315.
Skubitz
A P
AP
D000595:Amino Acid Sequence; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects*; D007797:Laminin / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*
D007797:Laminin; D010446:Peptide Fragments; D002918:Chymotrypsin