S100A11, S100A10, annexin I, desmosomal proteins, small proline-rich proteins, plasminogen activator inhibitor-2, and involucrin are components of the cornified envelope of cultured human epidermal keratinocytes.
The cornified envelope (CE) is an insoluble sheath of epsilon-(gamma-glutamyl)lysine cross-linked protein, which is deposited beneath the plasma membrane during keratinocyte terminal differentiation. We have probed the structure of the CE by proteolytic cleavage of purified CE fragments isolated from CEs formed spontaneously in cell culture. CNBr digestion, followed by trypsin and then proteinase K treatment released 25%, 42%, and 18%, respectively, of the CE protein. Purification and sequencing of released peptides has identified two novel CE precursors, S100A11 (S100C, calgizzarin) and S100A10 (calpactin light chain). We also sequenced peptides derived from annexin I and plasminogen activator inhibitor 2, two putative envelope precursors, as well as portions of the well established CE precursor proteins SPR1A, SPR1B, and involucrin. Many desmosomal components were identified (desmoglein 3, desmocolin A/B, desmoplakin I, plakoglobin, and plakophilin), indicating that desmosomes become cross-linked into the CE. Fragments derived from envoplakin, the recently sequenced 210-kDa membranous CE precursor protein, which also appears to be a desmosomal component, were also identified. Analysis of the pattern of peptide release following the sequential digestion indicates that S100A11 is anchored to the envelope via Gln102 and/or Lys103 at the carboxyl terminus and at Lys3, Lys23, and/or Gln22 in the amino terminus. A similar type of analysis indicates that small proline-rich proteins 1A and 1B (SPR1A and SPR1B) become cross-linked at the amino terminus (residues 1-23) and the carboxyl terminus (residues 86-89). No loricrin, cystatin A, or elafin peptides were detected.
Robinson
N A
NA
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Lapic
S
S
Welter
J F
JF
Eckert
R L
RL
D000595:Amino Acid Sequence; D017305:Annexin A1 / Q000032:analysis* / Q000737:chemistry; D017302:Annexins / Q000032:analysis* / Q000737:chemistry; D002454:Cell Differentiation; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D000078404:Epidermal Cells; D004817:Epidermis / Q000737:chemistry*; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry* / Q000166:cytology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis; D017396:Plasminogen Activator Inhibitor 2 / Q000032:analysis* / Q000737:chemistry; D055232:Proline-Rich Protein Domains; D011498:Protein Precursors / Q000032:analysis* / Q000737:chemistry; D009418:S100 Proteins / Q000032:analysis* / Q000737:chemistry
D017305:Annexin A1; D017302:Annexins; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D010455:Peptides; D010750:Phosphoproteins; D017396:Plasminogen Activator Inhibitor 2; D011498:Protein Precursors; D009418:S100 Proteins; C072337:S100A11 protein, human; C032253:involucrin