Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no α or β structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross- linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross- linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.
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